Polypeptides having dipeptidyl aminopeptidase activity and nucleic acids encoding same

ABSTRACT

The present invention relates to isolated polypeptides having dipeptidyl aminopeptidase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/857,884 filed on May 16, 1997, now abandoned, and claim benefit fromprovisional U.S. application Ser. No. 60/062,892 filed on Oct. 20, 1997,which applications are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having dipeptidylaminopeptidase activity and isolated nucleic acid sequences encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences as well asmethods for producing the polypeptides. The present invention furtherrelates to methods of obtaining protein hydrolysates useful as flavourimproving agents.

2. Description of the Related Art

Various food products, e.g., soups, sauces and seasonings, containflavoring agents obtained by hydrolysis of proteinaceous materials. Thishydrolysis is conventionally accomplished using strong hydrochloricacid, followed by neutralization with sodium hydroxide. However, suchchemical hydrolysis leads to severe degradation of the amino acidsobtained during the hydrolysis, and also to hazardous byproducts formedin the course of this chemical reaction. Increasing concern over the useof flavoring agents obtained by chemical hydrolysis has led to thedevelopment of enzymatic hydrolysis processes.

Enzymatic hydrolysis processes of proteinaceous materials aim atobtaining a high degree of hydrolysis (DH), and this is usually attainedusing a complex of unspecific acting proteolytic enzymes (i.e.,unspecific-acting endo- and exo-peptidases). For example, WO 94/25580describes a method for hydrolyzing proteins by use of an unspecificacting enzyme preparation obtained from Aspergillus oryzae. Specificacting proteolytic enzymes have not been used for this purpose becausesuch enzymes only lead to an inadequate degree of hydrolysis.

Polypeptides having dipeptidyl aminopeptidase activity catalyze theremoval of dipeptides from the N-terminus of peptides, polypeptides, andproteins. Such polypeptides are classified under the EnzymeClassification Number E.C. 3.4.14.—of the International Union ofBiochemistry and Molecular Biology.

Beauvais et al. (1997, Journal of Biological Chemistry 272: 6238-6244)disclose a dipeptidyl-peptidase from Aspergillus fumigatus which has amolecular weight of 88 kDa by SDS-PAGE and a substrate specificitylimited to the hydrolysis of X-Ala, His-Ser, and Ser-Tyr dipeptides at aneutral pH optimum. Tachi et al. (1992, Phytochemistry 31: 3707-3709)disclose an X-prolyl dipeptidyl aminopeptidase from Aspergillus oryzaewhich has a molecular weight of 145 kDa by SDS-PAGE and a substratespecificity toward the peptide bond at the carboxyl site of a prolineresidue in the penultimate position of N-terminal free dipeptides andamides at a neutral pH optimum.

The production of protein hydrolysates with desirable organolepticproperties and high degrees of hydrolysis generally requires the use ofa mixture of peptidase activities. It would be desirable to provide asingle component peptidase enzyme which has activity useful forimproving the organoleptic properties and degree of hydrolysis ofprotein hydrolysates used in food products either alone or incombination with other enzymes.

It is an object of the present invention to provide improvedpolypeptides having dipeptidyl aminopeptidase activity as well asmethods for obtaining protein hydrolysates with desirable organolepticqualities and high degrees of hydrolysis.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having dipeptidylaminopeptidase activity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 50%identity with the amino acid sequence of SEQ ID NO:2;

(b) a polypeptide encoded by a nucleic acid sequence which hybridizesunder medium stringency conditions with (i) the nucleic acid sequence ofSEQ ID NO: 1, (ii) its complementary strand, or (iii) a subsequencethereof;

(c) an allelic variant of (a) or (b); and

(d) a fragment of (a), (b), or (c), wherein the fragment has dipeptidylaminopeptidase activity; and

(e) a polypeptide having dipeptidyl aminopeptidase activity withphysicochemical properties of (i) a pH optimum in the range of fromabout pH 4.4 to about pH 9.8 determined after incubation for 5 minutesat ambient temperature in the presence of Ala-Pro-para-nitroanilide;(ii) a temperature stability of 90% or more, relative to initialactivity, at pH 7.5 determined after incubation for 20 minutes at 65° C.in the absence of substrate; and (iii) an activity towardsXaa-Pro-para-nitroanilide or Xaa-Ala-para-nitroanilide wherein Xaa isselected from the group consisting of Ala, Arg, Asp, Gly, and Val.

The present invention also relates to isolated nucleic acid sequencesencoding the polypeptides and to nucleic acid constructs, vectors, andhost cells comprising the nucleic acid sequences as well as methods forproducing the polypeptides.

The present invention also relates to methods for obtaining hydrolysatesfrom proteinaceous substrates which comprise subjecting theproteinaceous material to a polypeptide with dipeptidyl aminopeptidaseactivity alone or in combination with an endopeptidase, and tohydrolysates obtained from the method.

The present invention also relates to methods for obtaining from aproteinaceous substrate a hydrolysate enriched in free glutamic acidand/or peptide bound glutamic acid residues, which methods comprisesubjecting the substrate to a deamidation process and to the action of apolypeptide having dipeptidyl aminopeptidase activity.

The present invention further relates to flavor-improving compositionscomprising a polypeptide with dipeptidyl aminopeptidase activity.

In a final aspect, the methods of the invention may be used in foodrelated applications to improve flavor, such as baking. Alternatively,flavor improvement in foods may be achieved by the addition ofhydrolysates obtained by the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the nucleic acid sequence and the deduced aminoacid sequence of an Aspergillus oryzae ATCC 20386 dipeptidylaminopeptidase (SEQ ID NOS:1 and 2, respectively).

FIG. 2 shows a restriction map of pDM181.

FIG. 3 shows a restriction map of pMWR54.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides having DipeptidylAminopeptidase Activity

The term “dipeptidyl aminopeptidase activity” is defined herein as apeptidase activity which cleaves dipeptides from the N-terminal end of apeptide, polypeptide, or protein sequence. Defined in a general manner,the dipeptidyl aminopeptidase is capable of cleaving the dipeptide XYfrom the unsubstituted N-terminal amino group of a peptide, polypeptide,or protein, wherein X or Y may represent any amino acid residue selectedfrom the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val, but atleast Ala, Arg, Asp, Gly, and/or Val. All of X and Y may be different oridentical. It will be understood that the isolated polypeptides havingdipeptidyl aminopeptidase activity of the present invention may beunspecific as to the amino acid sequence of the dipeptide to be cleaved.

In a first embodiment, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to the amino acid sequence of SEQ ID NO:2 of at least about50%, preferably at least about 60%, preferably at least about 70%, morepreferably at least about 80%, even more preferably at least about 90%,most preferably at least about 95%, and even most preferably at leastabout 97%, which have dipeptidyl aminopeptidase activity (hereinafter“homologous polypeptides”). In a preferred embodiment, the homologouspolypeptides have an amino acid sequence which differs by five aminoacids, preferably by four amino acids, more preferably by three aminoacids, even more preferably by two amino acids, and most preferably byone amino acid from the amino acid sequence of SEQ ID NO:2. For purposesof the present invention, the degree of identity between two amino acidsequences is determined by the Clustal method (Higgins, 1989, CABIOS 5:151-153) with an identity table, a gap penalty of 10, and a gap lengthpenalty of 10.

Preferably, the polypeptides of the present invention comprise the aminoacid sequence of SEQ ID NO:2 or an allelic variant; and a fragmentthereof, wherein the fragment has dipeptidyl aminopeptidase activity. Ina more preferred embodiment, the polypeptides of the present inventioncomprise the amino acid sequence of SEQ ID NO:2. In another preferredembodiment, the polypeptide of the present invention has the amino acidsequence of SEQ ID NO:2 or a fragment thereof, wherein the fragment hasdipeptidyl aminopeptidase activity. A fragment of SEQ ID NO:2 is apolypeptide having one or more amino acids deleted from the amino and/orcarboxy terminus of this amino acid sequence. In a most preferredembodiment, the polypeptide has the amino acid sequence of SEQ ID NO:2.

Preferably, a fragment contains at least 455 amino acid residues, morepreferably at least 555 amino acid residues, and most preferably atleast 655 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chomosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. The term allelic variant is also used to denote aprotein encoded by an allelic variant of a gene.

The amino acid sequences of the homologous polypeptides may differ fromthe amino acid sequence of SEQ ID NO:2 by an insertion or deletion ofone or more amino acid residues and/or the substitution of one or moreamino acid residues by different amino acid residues. Preferably, aminoacid changes are of a minor nature, that is conservative amino acidsubstitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (such as arginine, lysine and histidine), acidic amino acids(such as glutamic acid and aspartic acid), polar amino acids (such asglutamine and asparagine), hydrophobic amino acids (such as leucine,isoleucine and valine), aromatic amino acids (such as phenylalanine,tryptophan and tyrosine), and small amino acids (such as glycine,alanine, serine, threonine and methionine). Amino acid substitutionswhich do not generally alter the specific activity are known in the artand are described, for example, by H. Neurath and R. L. Hill, 1979, In,The Proteins, Academic Press, New York. The most commonly occurringexchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly as well as these in reverse.

In a second embodiment, the present invention relates to isolatedpolypeptides having dipeptidyl aminopeptidase activity which are encodedby nucleic acid sequences which hybridize under low stringencyconditions, more preferably medium stringency conditions, and mostpreferably high stringency conditions, with an oligonucleotide probewhich hybridizes under the same conditions with the nucleic acidsequence of SEQ ID NO:1 or its complementary strand (J. Sambrook, E. F.Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.); or allelic variants and fragmentsof the polypeptides, wherein the fragments have dipeptidylaminopeptidase activity.

Hybridization indicates that the nucleic acid sequence hybridizes to theoligonucleotide probe corresponding to the polypeptide encoding part ofthe nucleic acid sequence shown in SEQ ID NO:1, under low to highstringency conditions (i.e., prehybridization and hybridization at 42°C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon spermDNA, and either 25, 35 or 50% formamide for low, medium and highstringencies, respectively), following standard Southern blottingprocedures.

The amino acid sequence of SEQ ID NO:2 or a partial sequence thereof maybe used to design an oligonucleotide probe, or a nucleic acid sequenceencoding a polypeptide of the present invention, such as the nucleicacid sequence of SEQ ID NO:1, or a subsequence thereof, may be used toidentify and clone DNA encoding polypeptides having dipeptidylaminopeptidase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, preferably at least 25, and more preferably at least 40nucleotides in length. Longer probes can also be used. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

Thus, a genomic, cDNA or combinatorial chemical library prepared fromsuch other organisms may be screened for DNA which hybridizes with theprobes described above and which encodes a polypeptide having dipeptidylaminopeptidase activity. Genomic or other DNA from such other organismsmay be separated by agarose or polyacrylarmide gel electrophoresis, orother separation techniques. DNA from the libraries or the separated DNAmay be transferred to and immobilized on nitrocellulose or othersuitable carrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, the carrier material is used in a Southernblot in which the carrier material is finally washed three times for 30minutes each using 2×SSC, 0.2% SDS preferably at least 50° C., morepreferably at least 55° C., more preferably at least 60° C., morepreferably at least 65° C., even more preferably at least 70° C., andmost preferably at least 75° C. Molecules to which the oligonucleotideprobe hybridizes under these conditions are detected using X-ray film.

In a third embodiment, the present invention relates to isolatedpolypeptides having the following physicochemical properties: (a) a pHoptimum in the range of from about pH 4.4 to about pH 9.8 determinedafter incubation for 5 minutes at ambient temperature in the presence ofAla-Pro-para-nitroanilide; (b) a temperature stability of 90% or more,relative to initial activity, at pH 7.5 determined after incubation for20 minutes at 65° C. in the absence of substrate; and (c) an activitytowards Xaa-Pro-para-nitroanilide or Xaa-Ala-para-nitroanilide whereinXaa is selected from the group consisting of Ala, Arg, Asp, Gly, andVal. The polypeptides of the present invention also have the ability tohydrolyze other substrates.

In a preferred embodiment, the pH optimum in the range of from about pH4.4 to about pH 9.8, more preferably in the range of from about pH 5.8to about pH 9.8, and most preferably in the range of from about pH 7.5to about pH 9.3 determined after incubation for 5 minutes at ambienttemperature in the presence of Ala-Pro-para-nitroanilide.

In another preferred embodiment, a polypeptide of the present inventionacts synergistically with an aminopeptidase to hydrolyze anotherpolypeptide. The term “acts synergistically with an aminopeptidase tohydrolyze another polypeptide” is defined herein as the combination of adipeptidyl aminopeptidase and an aminopeptidase which increases at least5-fold, more preferably at least 10-fold, even more preferably at least25-fold, most preferably at least 50-fold, and even most preferably atleast 100-fold the hydrolysis of a peptide or a polypeptide relative toeither individual enzyme alone. The aminopeptidase may be anyaminopeptidase, but is preferably an aminopeptidase obtained fromAspergillus oryzae, and more preferably aminopeptidase I obtained fromAspergillus oryzae as described in WO 96/28542. The polypeptide ispreferably a tripeptide, but may be any larger peptide or protein.

In a fourth embodiment, the present invention relates to isolatedpolypeptides having immunochemical identity or partial immunochemicalidentity to the polypeptide having the amino acid sequence of SEQ IDNO:2. The immunochemical properties are determined by immunologicalcross-reaction identity tests by the well-known Ouchterlony doubleimmunodiffusion procedure. Specifically, an antiserum containingantibodies which are immunoreactive or bind to epitopes of thepolypeptide having the amino acid sequence of SEQ ID NO:2 are preparedby immunizing rabbits (or other rodents) according to the proceduredescribed by Harboe and Ingild, In N. H. Axelsen, J. Krøll, and B.Weeks, editors, A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone andThorpe, Immunochemistry in Practice, Blackwell Scientific Publications,1982 (more specifically pages 27-31). A polypeptide havingimmunochemical identity is a polypeptide which reacts with the antiserumin an identical fashion such as total fusion of precipitates, identicalprecipitate morphology, and/or identical electrophoretic mobility usinga specific immunochemical technique. A further explanation ofimmunochemical identity is described by Axelsen, Bock, and Krøll, In N.H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual of QuantitativeImmunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter10. A polypeptide having partial immunochemical identity is apolypeptide which reacts with the antiserum in a partially identicalfashion such as partial fusion of precipitates, partially identicalprecipitate morphology, and/or partially identical electrophoreticmobility using a specific immunochemical technique. A furtherexplanation of partial immunochemical identity is described by Bock andAxelsen, In N. H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual ofQuantitative Immunoelectrophoresis, Blackwell Scientific Publications,1973, Chapter 11.

Polypeptides encoded by nucleic acid sequences which hybridize with anoligonucleotide probe which hybridizes with the nucleic acid sequence ofSEQ ID NO:1, its complementary strand, or allelic variants andsubsequences of SEQ ID NO:1; allelic variants and fragments of thepolypeptides; or the homologous polypeptides and polypeptides havingidentical or partially identical immunological properties may beobtained from microorganisms of any genus.

In a preferred embodiment, these polypeptides may be obtained from abacterial source.

For example, these polypeptides may be obtained from a gram positivebacterium such as a Bacillus strain, e.g., Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus stearothermophilus,Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain,e.g., Streptomyces lividans or Streptomyces murinus; or from a gramnegative bacterium, e.g., E. coli or Pseudomonas sp.

The polypeptides may be obtained from a fungal source, and morepreferably from a yeast strain such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or a filamentousfungal strain such as an Acremonium, Aspergillus, Aureobasidium,Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia,Tolypociadium, or Trichoderma strain.

In a preferred embodiment, the polypeptides are obtained from aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis strain.

In another preferred embodiment, the polypeptides are obtained from aFusarium bactridioides, Fusarium cereals, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium solani, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride strain.

The polypeptides of the present invention are preferably obtained fromspecies of Aspergillus including, but not limited to, Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, or Aspergillusoryzae.

In a more preferred embodiment, a polypeptide of the present inventionis obtained from an Aspergillus oryzae strain, and most preferably fromAspergillus oryzae ATCC 20386 or a mutant strain thereof, e.g., thepolypeptide with the amino acid sequence of SEQ ID NO:2.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents. The polypeptides of the presentinvention may also be obtained from microorganisms which are synonyms ofAspergillus as defined by Raper, K. D. and Fennel, D. I., 1965, TheGenus Aspergillus, The Wilkins Company, Baltimore. Aspergilli aremitosporic fungi characterized by an aspergillum conprised of aconidiospore stipe with no known teleomorphic states terminating in avesicle, which in turn bears one or two layers of synchronously formedspecialized cells, variously referred to as sterigmata or phialides, andasexually formed spores referred to as conidia. Known teleomorphs ofAspergillus include Eurotium, Neosartorya, and Emericella. Strains ofAspergillus and teleomorphs thereof are readily accessible to the publicin a number of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding the polypeptide has been detected withthe probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

For purposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the polypeptideis produced by the source or by a cell in which a gene from the sourcehas been inserted.

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other non-dipeptidyl aminopeptidase polypeptides,e.g., at least about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequenceswhich encode a polypeptide of the present invention. In a preferredembodiment, the nucleic acid sequence encodes a polypeptide obtainedfrom Aspergillus, e.g., Aspergillus oryzae, and in a more preferredembodiment, the nucleic acid sequence is obtained from Aspergillusoryzae ATCC 20386, e.g., the nucleic acid sequence of SEQ ID NO:1. Inanother more preferred embodiment, the nucleic acid sequence is thesequence contained in plasmid pMWR52 which is contained in Escherichiacoli NRRL B-21682. The present invention also encompasses nucleic acidsequences which encode a polypeptide having the amino acid sequence ofSEQ ID NO:2, which differ from SEQ ID NO:1 by virtue of the degeneracyof the genetic code. The present invention also relates to subsequencesof SEQ ID NO:1 which encode fragments of SEQ ID NO:2 which havedipeptidyl aminopeptidase activity. A subsequence of SEQ ID NO:1 is anucleic acid sequence encompassed by SEQ ID NO:1 except that one or morenucleotides from the 5′ end and/or 3′ end have been deleted. Preferably,a subsequence contains at least 990 nucleotides, more preferably atleast 1140 nucleotides, and most preferably at least 1290 nucleotides.

The nucleic acid sequences may be obtained from microorganisms which aretaxonomic equivalents of Aspergillus as defined by Raper, K. D. andFennel, D. I., 1965, supra., regardless of the species name by whichthey are known.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Aspergillus, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

The present invention also relates to nucleic acid sequences which havea degree of homology to the nucleic acid sequence of SEQ ID NO:1 of atleast about 50%, preferably about 60%, preferably about 70%, preferablyabout 80%, more preferably about 90%, even more preferably about 95%,and most preferably about 97% homology, which encode an activepolypeptide. For purposes of the present invention, the degree ofhomology between two nucleic acid sequences is determined by the Clustalmethod (Higgins, 1989, supra) with an identity table, a gap penalty of10, and a gap length penalty of 10.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source. For example, itmay be of interest to synthesize variants of the polypeptide where thevariants differ in specific activity, thermostability, pH optimum, orthe like using, e.g., site-directed mutagenesis. The analogous sequencemay be constructed on the basis of the nucleic acid sequence presentedas the polypeptide encoding part of SEQ ID NO:1, e.g., a subsequencethereof, and/or by introduction of nucleotide substitutions which do notgive rise to another amino acid sequence of the polypeptide encoded bythe nucleic acid sequence, but which corresponds to the codon usage ofthe host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor dipeptidyl aminopeptidase activity to identify amino acid residuesthat are critical to the activity of the molecule. Sites ofsubstrate-enzyme interaction can also be determined by analysis of thethree-dimensional structure as determined by such techniques as nuclearmagnetic resonance analysis, crystallography or photoaffinity labelling(see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al.,1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992,FEBS Letters 309: 59-64).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleic acid sequence (or aportion thereof) encoding another polypeptide to a nucleic acid sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize underlow stringency conditions, more preferably medium stringency conditions,and most preferably high stringency conditions, with an oligonucleotideprobe which hybridizes under the same conditions with the nucleic acidsequence of SEQ ID NO:1 or its complementary strand; or allelic variantsand subsequences thereof (Sambrook et al., 1989, supra).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” as definedherein is a sequence which is transcribed into mRNA and translated intoa polypeptide of the present invention. The boundaries of the codingsequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′ end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′ end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceencoding a polypeptide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying nucleic acid sequences utilizing cloningmethods are well known in the art.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, a polyadenylationsequence, a propeptide sequence, a promoter, a signal sequence, and atranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the production of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80: 21-25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (U.S. Pat. No.4,288,627), and mutant, truncated, and hybrid promoters thereof.Particularly preferred promoters for use in filamentous fungal hostcells are the TAKA amylase, NA2-tpi (a hybrid of the promoters from thegenes encoding Aspergillus niger neutral alpha-amylase and Aspergillusoryzae triose phosphate isomerase), and glaA promoters.

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL 1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8: 423-488. In a mammalian host cell, useful promotersinclude viral promoters such as those from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus, bovine papilloma virus (BPV), and humancytomegalovirus (CMV).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.Terminator sequences are well known in the art for mammalian host cells.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence which is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes encoding Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from theSaccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomycescerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiaealpha-factor, and the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Molecular Cellular Biology 15: 5983-5990. Polyadenylationsequences are well known in the art for mammalian host cells.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not normallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to obtain enhanced secretion of thepolypeptide. The signal peptide coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, a lipase orproteinase gene from a Rhizomucor species, the gene for the alpha-factorfrom Saccharomyces cerevisiae, an amylase or a protease gene from aBacillus species, or the calf preprochymosin gene. However, any signalpeptide coding region which directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used in the presentinvention.

An effective signal peptide coding region for bacterial host cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), or theBacillus subtilis PrsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57: 109-137.

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from the Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene,Rhizomucor miehei aspartic proteinase gene, Humicola lanuginosacellulase gene, or Humicola lanuginosa lipase gene.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, the Rhizomucor miehei aspartic proteinase gene, or theMyceliophthora thermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

The nucleic acid constructs of the present invention may also compriseone or more nucleic acid sequences which encode one or more factors thatare advantageous for directing the expression of the polypeptide, e.g.,a transcriptional activator (e.g., a trans-acting factor), a chaperone,and a processing protease. Any factor that is functional in the hostcell of choice may be used in the present invention. The nucleic acidsencoding one or more of these factors are not necessarily in tandem withthe nucleic acid sequence encoding the polypeptide.

A transcriptional activator is a protein which activates transcriptionof a nucleic acid sequence encoding a polypeptide (Kudla et al., 1990,EMBO Journal 9: 1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244; Verdier, 1990, Yeast 6: 271-297). The nucleic acid sequenceencoding an activator may be obtained from the genes encoding Bacillusstearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activatorprotein 1 (hap1), Saccharomyces cerevisiae galactose metabolizingprotein 4 (gal4), Aspergillus nidulans amnonia regulation protein(areA), and Aspergillus oryzae alpha-amylase activator (amyR). Forfurther examples, see Verdier, 1990, supra and MacKenzie et al., 1993,Journal of General Microbiology 139: 2295-2307.

A chaperone is a protein which assists another polypeptide in foldingproperly (Hartl et al., 1994, TIBS 19: 20-25; Bergeron et al., 1994,TIBS 19: 124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189; Craig, 1993, Science 260: 1902-1903; Gething and Sambrook,1992, Nature 355: 33-45; Puig and Gilbert, 1994, Journal of BiologicalChemistry 269: 7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157; Robinson et al., 1994, Bio/Technology 1: 381-384; Jacobs etal., 1993, Molecular Microbiology 8: 957-966). The nucleic acid sequenceencoding a chaperone may be obtained from the genes encoding Bacillussubtilis GroE proteins, Bacillus subtilis PrsA, Aspergillus oryzaeprotein disulphide isomerase, Saccharomyces cerevisiae calnexin,Saccharomyces cerevisiae BiP/GRP78, and Saccharomyces cerevisiae Hsp70.For further examples, see Gething and Sambrook, 1992, supra, and Hartlet al., 1994, supra.

A processing protease is a protease that cleaves a propeptide togenerate a mature biochemically active polypeptide (Enderlin andOgrydziak, 1994, Yeast 10: 67-79; Fuller et al., 1989, Proceedings ofthe National Academy of Sciences USA 86: 1434-1438; Julius et al., 1984,Cell 37: 1075-1089; Julius et al., 1983, Cell 32: 839-852; U.S. Pat. No.5,702,934). The nucleic acid sequence encoding a processing protease maybe obtained from the genes encoding Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, Yarrowia lipolyticadibasic processing endoprotease (xpr6), and Fusarium oxysporummetalloprotease (p45 gene).

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more targeting sequences canbe used. For example, a circular plasmid or DNA fragment preferablyemploys a single targeting sequence, while a linear plasmid or DNAfragment preferably employs two targeting sequences.

The regulatory sequence of the construct can be comprised of one or morepromoters, enhancers, scaffold-attachment regions or matrix attachmentsites, negative regulatory elements, transcription binding sites, orcombinations of these sequences.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′ non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon.

Typically, the first exon lies 5′ of the second exon, and thesplice-donor site overlapping and flanking the first exon on its 3′ siderecognizes a splice-acceptor site flanking the second exon on the 5′side of the second exon. A splice-acceptor site, like a splice-donorsite, is a sequence which directs the splicing of one exon to anotherexon. Acting in conjunction with a splice-donor site, the splicingapparatus uses a splice-acceptor site to effect the removal of anintron.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids. The vector may be anautonomously replicating vector, i.e., a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome. The vector may contain anymeans for assuring self-replication. Alternatively, the vector may beone which, when introduced into the host cell, is integrated into thegenome and replicated together with the chromosome(s) into which it hasbeen integrated. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids which together contain the total DNA tobe introduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers formammalian cells are the dihydrofolate reductase (dfhr), hygromycinphosphotransferase (hygB), aminoglycoside phosphotransferase II, andphleomycin resistance genes. Suitable markers for yeast host cells areADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker foruse in a filamentous fungal host cell may be selected from the groupincluding, but not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents from other species.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus. Furthermore, selection may be accomplishedby co-transformation, e.g., as described in WO 91/17243, where theselectable marker is on a separate vector.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB 110, pE 194, pTA 1060, andpAMβ1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes its functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1433).

More than one copy of a nucleic acid sequence encoding a polypeptide ofthe present invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by culturing the cells in the presence of theappropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. The term “host cell”encompasses any progeny of a parent cell which is not identical to theparent cell due to mutations that occur during replication.

A vector comprising a nucleic acid sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vector.Integration is generally considered to be an advantage as the nucleicacid sequence is more likely to be stably maintained in the cell.Integration of the vector into the host chromosome may occur byhomologous or non-homologous recombination as described above.

The choice of a host cell will to a large extent depend upon the geneencoding the polypeptide and its source. The host cell may be aunicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote. Useful unicellular cells are bacterialcells such as gram positive bacteria including, but not limited to, aBacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacilluscoagulans, Bacillus lautus, Bacillus tentus, Bacillus licheniformis,Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, andBacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli and Pseudomonas sp. In a preferred embodiment, the bacterial hostcell is a Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus, or Bacillus subtilis cell. The introduction of avector into a bacterial host cell may, for instance, be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, MolecularGeneral Genetics 168: 111-115), by using competent cells (see, e.g.,Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnauand Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221),by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987,Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian cell, an insectcell, a plant cell or a fungal cell. Useful mammalian cells includeChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, COS cells, or any number of other immortalized cell linesavailable, e.g., from the American Type Culture Collection.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al, 1995, supra, page 171) and all mitosporicfungi (Hawksworth et al., 1995, supra). Representative groups ofAscomycota include, e.g., Neurospora, Eupenicillium (=Penicillium),Emericella (=Aspergillus), Eurotium (=Aspergillus), and the true yeastslisted below. Examples of Basidiomycota include mushrooms, rusts, andsmuts. Representative groups of Chytridiomycota include, e.g.,Allomyces, Blastocladiella, Coelomomyces, and aquatic fungi.Representative groups of Oomycota include, e.g., Saprolegniomycetousaquatic fungi (water molds) such as Achlya. Examples of mitosporic fungiinclude Aspergillus, Penicillium, Candida, and Alternaria.Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Kluyveromyces, Pichia, andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeast belonging to the Fungi Imperfecti are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biologyof yeast and manipulation of yeast genetics are well known in the art(see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A. O. M., editors, 2nd edition, 3s 1987; The Yeasts,Rose, A. H., and Harrison, J. S., editors, 2nd edition, 1987; and TheMolecular Biology of the Yeast Saccharomyces, Strathern et al., editors,1981).

In an even more preferred embodiment, the yeast host cell is a cell of aspecies of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative. In a more preferred embodiment, the filamentous fungalhost cell is a cell of a species of, but not limited to, Acremonium,Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,Penicillium, Thielavia, Tolypocladium, and Trichoderma.

In an even more preferred embodiment, the filamentous fungal host cellis an Aspergillus cell. In another even more preferred embodiment, thefilamentous fungal host cell is an Acremonium cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Fusariumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Humicola cell. In another even more preferred embodiment,the filamentous fungal host cell is a Mucor cell. In another even morepreferred embodiment, the filamentous fungal host cell is aMyceliophthora cell. In another even more preferred embodiment, thefilamentous fungal host cell is a Neurospora cell. In another even morepreferred embodiment, the filamentous fungal host cell is a Penicilliumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Thielavia cell. In another even more preferredembodiment, the filamentous fungal host cell is a Tolypocladium cell. Inanother even more preferred embodiment, the filamentous fungal host cellis a Trichoderma cell.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium solani, Fusarium sporotrichioides Fusarium sulphureum, Fusariumtorulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In aneven most preferred embodiment, the filamentous fungal parent cell is aFusarium venenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens orHumicola lanuginosa cell. In another most preferred embodiment, thefilamentous fungal host cell is a Mucor miehei cell. In another mostpreferred embodiment, the filamentous fungal host cell is aMyceliophthora thermophilum cell. In another most preferred embodiment,the filamentous fungal host cell is a Neurospora crassa cell. In anothermost preferred embodiment, the filamentous fungal host cell is aPenicillium purpurogenum cell. In another most preferred embodiment, thefilamentous fungal host cell is a Thielavia terrestris cell. In anothermost preferred embodiment, the Trichoderma cell is a Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474 Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920. Mammalian cells may betransformed by direct uptake using the calcium phosphate precipitationmethod of Graham and Van der Eb (1978, Virology 52: 546).

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide. Preferably, the strain is of the genusAspergillus.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulatory sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670. Geneactivation technology is based on activating a gene which is normallyunexpressed in a cell or increasing expression of a gene which isexpressed at very low levels in a cell. Gene activation technologyincludes methods of inserting an exogenous DNA construct containing aregulatory sequence, an exon, and/or a splice donor site into thegenomic DNA of a cell in such a manner that the insertion results in theproduction of a new transcription unit in which the regulatory sequence,the exon, and/or the splice donor site are operably linked to andactivate expression of the endogenous gene.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art (see, e.g., references forbacteria and yeast; Bennett, J. W. and LaSure, L., editors, More GeneManipulations in Fungi, Academic Press, California, 1991). Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide. Procedures for determiningdipeptidyl aminopeptidase activity are known in the art and include,e.g., measuring the initial rate of hydrolysis of a p-nitroanilide at405 nm.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing, differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see,e.g.,Protein Purification, J. -C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Removal or Reduction of Dipeptidyl Aminopeptidase Activity

The present invention also relates to methods for producing a mutantcell of a parent cell, which comprises disrupting or deleting a nucleicacid sequence encoding the polypeptide or a control sequence thereof,which results in the mutant cell producing less of the polypeptide thanthe parent cell.

The construction of strains which have reduced dipeptidyl aminopeptidaseactivity may be conveniently accomplished by modification orinactivation of a nucleic acid sequence necessary for expression of thepolypeptide having dipeptidyl aminopeptidase activity in the cell. Thenucleic acid sequence to be modified or inactivated may be, for example,a nucleic acid sequence encoding the polypeptide or a part thereofessential for exhibiting dipeptidyl aminopeptidase activity, or thenucleic acid sequence may have a regulatory function required for theexpression of the polypeptide from the coding sequence of the nucleicacid sequence. An example of such a regulatory or control sequence maybe a promoter sequence or a functional part thereof, i.e., a part whichis sufficient for affecting expression of the polypeptide. Other controlsequences for possible modification include, but are not limited to, aleader, a polyadenylation sequence, a propeptide sequence, a signalsequence, and a termination terminator.

Modification or inactivation of the nucleic acid sequence may beperformed by subjecting the cell to mutagenesis and selecting for cellsin which the dipeptidyl aminopeptidase producing capability has beenreduced or eliminated. The mutagenesis, which may be specific or random,may be performed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and selectingfor cells exhibiting reduced or no expression of dipeptidylaminopeptidase activity.

Modification or inactivation of production of a polypeptide of thepresent invention may be accomplished by introduction, substitution, orremoval of one or more nucleotides in the nucleic acid sequence encodingthe polypeptide or a regulatory element required for the transcriptionor translation thereof. For example, nucleotides may be inserted orremoved so as to result in the introduction of a stop codon, the removalof the start codon, or a change of the open reading frame. Suchmodification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art.

Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the nucleic acid sequence to bemodified, it is preferred that the modification be performed in vitro asexemplified below.

An example of a convenient way to inactivate or reduce production by ahost cell of choice is based on techniques of gene replacement or geneinterruption. For example, in the gene interruption method, a nucleicacid sequence corresponding to the endogenous gene or gene fragment ofinterest is mutagenized in vitro to produce a defective nucleic acidsequence which is then transformed into the host cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous gene or gene fragment. It may bedesirable that the defective gene or gene fragment also encodes a markerwhich may be used for selection of transformants in which the geneencoding the polypeptide has been modified or destroyed.

Alternatively, modification or inactivation of the nucleic acid sequenceencoding a polypeptide of the present invention may be performed byestablished anti-sense techniques using a nucleotide sequencecomplementary to the polypeptide encoding sequence. More specifically,production of the polypeptide by a cell may be reduced or eliminated byintroducing a nucleotide sequence complementary to the nucleic acidsequence encoding the polypeptide which may be transcribed in the celland is capable of hybridizing to the polypeptide mRNA produced in thecell. Under conditions allowing the complementary anti-sense nucleotidesequence to hybridize to the polypeptide mRNA, the amount of polypeptidetranslated is thus reduced or eliminated.

It is preferred that the cell to be modified in accordance with themethods of the present invention is of microbial origin, for example, afungal strain which is suitable for the production of desired proteinproducts, either homologous or heterologous to the cell.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleic acid sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide than the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising (a) culturing the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. In the present context, the term “heterologouspolypeptides” is defined herein as polypeptides which are not native tothe host cell, a native protein in which modifications have been made toalter the native sequence, or a native protein whose expression isquantitatively altered as a result of a manipulation of the host cell byrecombinant DNA techniques.

In a still further aspect, the present invention relates to a method forproducing a protein product essentially free of dipeptidylaminopeptidase activity by fermentation of a cell which produces both apolypeptide of the present invention as well as the protein product ofinterest.

The method comprises adding an effective amount of an agent capable ofinhibiting dipeptidyl aminopeptidase activity to the fermentation brotheither during or after the fermentation has been completed, recoveringthe product of interest from the fermentation broth, and optionallysubjecting the recovered product to further purification.

In a still further alternative aspect, the present invention relates toa method for producing a protein product essentially free of dipeptidylaminopeptidase activity, wherein the protein product of interest isencoded by a DNA sequence present in a cell encoding a polypeptide ofthe present invention. The method comprises cultivating the cell underconditions permitting the expression of the product, subjecting theresultant culture broth to a combined pH and temperature treatment so asto reduce the dipeptidyl aminopeptidase activity substantially, andrecovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with a dipeptidyl aminopeptidase inhibitor.

The combined pH and temperature treatment is preferably carried out at apH in the range of 9-11 and a temperature in the range of 40-75° C. fora sufficient period of time to attain the desired effect, wheretypically, 30 to 60 minutes is sufficient.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the dipeptidyl aminopeptidase activity. It is contemplated that acomplete removal of dipeptidyl aminopeptidase activity may be obtainedby use of these methods.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallydipeptidyl aminopeptidase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, a lipolytic enzyme, a proteolytic enzyme, a cellulytic enzyme,an oxidoreductase or a plant cell-wall degrading enzyme. Examples ofsuch enzymes include an aminopeptidase, an amylase, an amyloglucosidase,a carbohydrase, a carboxypeptidase, a catalase, a cellulase, achitinase, a cutinase, a cyclodextrin glycosyltransferase, adeoxyribonuclease, an esterase, a galactosidase, a beta-galactosidase, aglucoamylase, a glucose oxidase, a glucosidase, a haloperoxidase, ahemicellulase, an invertase, an isomerase, a laccase, a ligase, alipase, a lyase, a mannosidase, an oxidase, a pectinolytic enzyme, aperoxidase, a phytase, a phenoloxidase, a polyphenoloxidase, aproteolytic enzyme, a ribonuclease, a transferase, a transglutaminase,or a xylanase. The dipeptidyl aminopeptidase-deficient cells may also beused to express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from dipeptidyl aminopeptidase activity which isproduced by a method of the present invention.

Methods of Producing Protein Hydrolysates

The polypeptides of the present invention may be used in the productionof protein hydrolysates for enhancing the degree of hydrolysis andflavor development.

The present invention further relates to methods for using a polypeptideof the present invention in combination with an endopeptidase to producea high degree of hydrolysis of a protein-rich material. The methodcomprises treating of a proteinaceous substrate with the polypeptide andan endopeptidase. The substrate may be treated with the enzymesconcurrently or consecutively.

A polypeptide of the present invention is added to the proteinaceoussubstrate in an effective amount conventionally employed in proteinhydrolysis processes, preferably in the range of from about 0.1 to about100,000 dipeptidyl aminopeptidase units (DPAPU) per 100 g of protein,and more preferably in the range of from about 1 to about 10,000dipeptidyl aminopeptidase units per 100 g of protein. As defined herein,one dipeptidyl aminopeptidase Unit (DPAPU) is the amount of enyzmeneeded to release 1 micromole of p-nitroanilide per minute fromAla-Pro-p-nitroanilide (Sigma Chemical Co., St. Louis Mo.) underspecified conditions.

The endopeptidase may be obtained from a strain of Bacillus, preferablyBacillus licheniformis or Bacillus subtilis, a strain of Staphylococcus,preferably Staphylococcus aureus, a strain of Streptomyces, preferablyStreptomyces thermovularis or Streptomyces griseus, a strain ofActinomyces species, a strain of Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillusnidulans, Aspergillus niger, or Aspergillus oryzae, or a strain ofFusarium, preferably Fusarium venenatum.

The endopeptidase is added to the proteinaceous substrate in aneffective amount conventionally employed in protein hydrolysisprocesses, preferably in the range of from about 0.05 to about 15 AU/100g of protein, and more preferably from about 0.1 to about 8 AU/100 g ofprotein. One AU (Anson Unit) is defined as the amount of enzyme whichunder standard conditions (i.e., 25° C., pH 7.5 and 10 min. reactiontime) digests hemoglobin at an initial rate such that there is liberatedper minute an amount of TCA soluble product which gives the same colorwith phenol reagent as one milli-equivalent of tyrosine. The analyticalmethod AF 4/5 is available upon request from Novo Nordisk A/S, Denmark,which is incorporated herein by reference.

The enzymatic treatment, i.e., the incubation of the substrate with theenzyme preparations, may take place at any convenient temperature atwhich the enzyme preparation does not become inactivated, preferably inthe range of from about 20° C. to about 70° C. In accordance withestablished practice, the enzyme preparations may be suitablyinactivated by increasing the temperature of the incubation mixture to atemperature where the enzymes become inactivated, e.g., to above about70° C., or similarly by decreasing the pH of the incubation mixture to apoint where the enzymes become inactivated, e.g., below about 4.0.

Furthermore, the methods of the present invention result in enhancementof the degree of hydrolysis of a proteinaceous substrate. As usedherein, the degree of hydrolysis (DH) is the percentage of the totalnumber of amino bonds in a protein that has been hydrolyzed by aproteolytic enzyme.

In another aspect of the present invention, the hydrolysates have anincreased content of Ala, Arg, Asp, Gly, and/or Val, e.g., 1.1 timesgreater.

In another aspect of the present invention, a polypeptide of the presentinvention acts synergistically with an aminopeptidase to hydrolyze atripeptide where either enzyme activity alone does not hydrolyze thetripeptide. In a preferred embodiment, the aminopeptidase isaminopeptidase I obtained from Aspergillus oryzae as described in WO96/28542.

The present invention also relates to methods for obtaining a proteinhydrolysate enriched in free glutamic acid and/or peptide bound glutamicacid residues, which method comprises:

(a) subjecting the substrate to a deamidation process; and

(b) subjecting the substrate to the action of a polypeptide havingdipeptidyl aminopeptidase activity.

The two steps may be performed simultaneously, or the second step may beperformed subsequent to the first step.

These methods of the present invention produce protein hydrolysates ofexcellent flavor because glutamic acid (Glu), whether free or peptidebound, plays an important role in the flavor and palatability of proteinhydrolysates. These method also produce protein hydrolysates havingimproved functionality, in particular, improved solubility, improvedemulsifying properties, increased degree of hydrolysis, and improvedfoaming properties.

The conversion of amides (glutamine or asparagine) into charged acids(glutamic acid or aspartic acid) via the liberation of ammonia is knownas deamidation. Deamidation may take place as a non-enzymatic or as anenzymatic deamidation process.

In a preferred embodiment, the deamidation is carried out as anenzymatic deamidation process, e.g., by subjecting the substrate to atransglutaminase and/or peptidoglutaminase.

The transglutaminase may be of any convenient source including mammals,see e.g., JP 1050382 and JP 5023182, including activated Factor XIII,see e.g., WO 93/15234; those derived from fish, see e.g., EP 555,649;and those obtained from microorganisms, see e.g., EP 379,606, WO96/06931 and WO 96/22366. In a preferred embodiment, thetransglutaminase is obtained from an Oomycete, including a strain ofPhytophthora, preferably Phytophthora cactorum, or a strain of Pythium,preferably Pythium irregulare, Pythium sp., Pythium intermedium, Pythiumultimum, or Pythium periilum (or Pythium periplocum). In anotherpreferred embodiment, the transglutaminase is of bacterial origin and isobtained from a strain of Bacillus, preferably Bacillus subtilis, astrain of Streptoverticillium, preferably Streptoverticilliummobaraensis, Streptoverticillium griseocarneum, or Streptoverticilliumcinnamoneum, and a strain of Streptomyces, preferably Streptomyceslydicus.

The peptidoglutaminase may be a peptidoglutaminase I(peptidyl-glutaminase; EC 3.5.1.43), or a peptidoglutaminase II(protein-glutamine glutaminase; EC 3.5.1.44), or any mixture thereof.The peptidoglutaminase may be obtained from a strain of Aspergillus,preferably Aspergillus japonicus, a strain of Bacillus, preferablyBacillus circulans, a strain of Cryptococcus, preferably Cryptococcusalbidus, or a strain of Debaryomyces, preferably Debaryomyces kloecheri.

The transglutaminase is added to the proteinaceous substrate in aneffective amount conventionally employed in deamidation processes,preferably in the range of from about 0.01 to about 5% (w/w), and morepreferably in the range of from about 0.1 to about 1% (w/w) of enzymepreparation relating to the amount of substrate.

The peptidoglutaminase is added to the proteinaceous substrate in aneffective amount conventionally employed in deamidation processes,preferably in the range of from about 0.01 to about 100,000 PGase Unitsper 100 g of substrate, and more preferably in the range of from about0.1 to about 10,000 PGase Units per 100 g of substrate.

The peptidoglutaminase activity may be determined according to theprocedure of Cedrangoro et al (1965, Enzymologia 29: 143). According tothis procedure, 0.5 ml of an enzyme sample, adjusted to pH 6.5 with I NNaOH, is charged into a small vessel. Then 1 ml of a borate pH 10.8buffer solution is added to the vessel. The discharged ammonia isabsorbed by 5 N sulphuric acid, and by use of Nessler's reagent themixture is allowed to form color which is measured at 420 nm. One PGaseunit is the amount of enzyme capable of producing 1 micromole of ammoniaper minute under these conditions.

Alternatively, the peptidoglutaminase activity may be determinedaccording to the procedure described in U.S. Pat No. 3,857,967 orExample 17 below.

In step (b) of the methods of the present invention, the substrate issubjected to a polypeptide of the present invention. A polypeptide ofthe present invention is added to the proteinaceous substrate in aneffective amount conventionally employed in protein hydrolysisprocesses, preferably in the range of from about 0.001 to about 0.5AU/100 g of substrate, more preferably in the range of from about 0.01to about 0.1 AU/100 g of substrate.

In another embodiment, the methods of the present invention may be usedto produce a hydrolysate enriched in free glutamic acid and/or peptidebound glutamic acid residues further comprise:

(c) subjecting the substrate to one or more unspecific acting endo-and/or exo-peptidase enzymes.

This step may take place simultaneously with steps (a) and (b), or mayfollow steps (a) and (b).

In a preferred embodiment, the unspecific acting endo- and/orexo-peptidase enzyme is obtained from a strain of Aspergillus,preferably Aspergillus niger, Aspergillus oryzae, or Aspergillus sojae,or a strain of Bacillus, preferably Bacillus amyloliquefaciens, Bacilluslentus, Bacillus licheniformis, or Bacillus subtilis.

The unspecific acting endo- and/or exo-peptidase enzyme is added to thesubstrate in an effective amount conventionally employed in proteinhydrolysis processes, preferably in the range of from about 0.05 toabout 15 CPU/100 g of substrate, and more preferably in the range offrom about 0.1 to about 5 CPU/100 g of substrate. One CPU (CaseinProtease Unit) is defined as the amount of enzyme liberating I micromoleof primary amino groups (determined by comparison with a serinestandard) per minute from casein under standard conditions, i.e.,incubation for 30 minutes at 25° C. and pH 9.5. The analytical method AF228/1, which is incorporated herein by reference, is available uponrequest from Novo Nordisk A/S, Bagsvaerd, Denmark.

Each enzymatic treatment may take place at any temperature at which theenzyme preparation does not become inactivated, preferably in the rangeof from about 20° C. to about 70° C. The enzyme preparation may then beinactivated by increasing the temperature, e.g., to above about 70° C.,or by decreasing the pH, e.g., below about 4.0.

The proteinaceous substrate used in the methods of the present inventionmay consist of intact proteins, prehydrolyzed proteins (i.e., peptides),or a mixture thereof. The proteinaceous substrate may be of vegetable oranimal origin. Preferably, the proteinaceous substrate is of vegetableorigin, e.g., soy protein, grain protein, e.g., wheat gluten, corngluten, barley, rye, oat, rice, zein, lupine, cotton seed protein, rapeseed protein, peanut, alfalfa protein, pea protein, fabaceous beanprotein, sesame seed protein, or sunflower. A proteinaceous substrate ofanimal origin may be whey protein, casein, meat proteins, fish protein,red blood cells, egg white, gelatin, or lactoalbumin.

The present invention also relates to protein hydrolysates produced bythese methods.

Other Uses

The present invention also relates to methods of deactivating enzymeswith a polypeptide of the present invention.

Furthermore, a polypeptide of the present invention may be useful for anumber of purposes in which a specific cleavage of peptide sequences isdesirable. For instance, some proteins or peptides are synthesized inthe form of inactive precursors comprising a number of additional aminoacid residues at the N-terminal of the mature protein. A polypeptide ofthe present invention could provide the necessary post-translationalprocessing to activate such precursor proteins.

Compositions

In a still further aspect, the present invention relates to polypeptidecompositions comprising a polypeptide of the present invention.Preferably, the compositions are enriched in a polypeptide of thepresent invention. In the present context, the term “enriched” indicatesthat the dipeptidyl aminopeptidase activity of the polypeptidecomposition has been increased, e.g., with an enrichment factor of 1.1.

The polypeptide composition may comprise a polypeptide of the inventionas the major enzymatic component, e.g., a mono-component polypeptidecomposition. Alternatively, the composition may comprise multipleenzymatic activities, such as an aminopeptidase, an amylase, acarbohydrase, a carboxypeptidase, a catalase, a cellulase, a chitinase,a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, anesterase, an alpha-galactosidase, a beta-galactosidase, a glucoamylase,an alpha-glucosidase, a beta-glucosidase, a haloperoxidase, aninvertase, a laccase, a lipase, a mannosidase, an oxidase, apectinolytic enzyme, a peptidoglutaminase, a peroxidase, a phytase, apolyphenoloxidase, a proteolytic enzyme, a ribonuclease, atransglutaminase, or a xylanase. The additional enzyme(s) may beproducible by means of a microorganism belonging to the genusAspergillus, preferably Aspergillus aculeatus, Aspergillus awamori,Aspergillus niger, or Aspergillus oryzae, or Trichoderma, Humicola,preferably Humicola insolens, or Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum.

In a preferred embodiment, the invention relates to a flavor-improvingcomposition comprising a polypeptide with dipeptidyl aminopeptidaseactivity and a carrier. Any suitable carrier known in the art may beused. In another preferred embodiment, the flavor-improving compositionfurther comprises an endopeptidase. In another preferred embodiment, theflavoring composition further comprises one or more unspecific-actingendo- and/or exo-peptidase enzymes. In another preferred embodiment, theflavoring composition further comprises one or more specific-actingendo- and/or exo-peptidase enzymes.

In a preferred embodiment, the specific acting proteolytic enzyme is anendopeptidase such as a glutamyl endopeptidase (EC 3.4.21.19); a lysylendopeptidase (EC 3.4.21.50); a leucyl endopeptidase (EC 3.4.21.57); aglycyl endopeptidase (EC 3.4.22.25); a prolyl endopeptidase (EC3.4.21.26); trypsin (EC 3.4.21.4) or a trypsin-like (lysine/argininespecific) endopeptidase; or a peptidyl-Asp metalloendopeptidase (EC3.4.24.33).

The glutamyl endopeptidase (EC 3.4.21.19) may preferably be obtainedfrom a Bacillus strain, in particular Bacillus licheniformis andBacillus subtilis, a Staphylococcus strain, in particular Staphylococcusaureus, a Streptomyces strain, in particular Streptomyces thermovulgarisand Streptomyces griseus, or a Actinomyces strain.

The lysyl endopeptidase (EC 3.4.21.50) may preferably be obtained from aAchromobacter strain, in particular Achromobacter lyticus, a Lysobacterstrain, in particular Lysobacter enzymogenes, or a Pseudomonas strain,in particular Pseudomonas aeruginosa.

The leucyl endopeptidase (EC 3.4.21.57) may be of plant origin.

The glycyl endopeptidase (EC 3.4.22.25) may preferably be obtained fromthe papaya plant (Carica papaya).

The prolyl endopeptidase (EC 3.4.21.26) may preferably be obtained froma Flavobacterium strain, or it may be of plant origin.

The trypsin-like endopeptidase may preferably be obtained from aFusarium strain, in particular Fusarium oxysporum, e.g., as described inWO 89/06270 or WO 94/25583.

The peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) may preferably beobtained from a Pseudomonas strain, in particular Pseudomonas fragi.

In another preferred embodiment, the specific acting proteolytic enzymeis an exo-peptidase that may act from either end of the peptide.

In a preferred embodiment, the specific acting proteolytic enzyme is anaminopeptidase such as a leucyl aminopeptidase (EC 3.4.11.1); or atripeptide aminopeptidase (EC 3.4.11.4).

In another preferred embodiment, the specific acting proteolytic enzymeis a carboxypeptidase such as a proline carboxypeptidase (EC 3.4.16.2);a carboxypeptidase A (EC 3.4.17.1); a carboxypeptidase B (EC 3.4.17.2);a carboxypeptidase C (EC 3.4.16.5); a carboxypeptidase D (EC 3.4.16.6);a lysine (arginine) carboxypeptidase (EC 3.4.17.3); a glycinecarboxypeptidase (EC 3.4.17.4); an alanine carboxypeptidase (EC3.4.17.6); a glutamate carboxypeptidase (EC 3.4.17.11); apeptidyl-dipeptidase A (EC 3.4.15.1); or a peptidyl-dipeptidase (EC3.4.15.5).

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. The polypeptide may be stabilized by methods known in theart.

The present invention also relates to food products, e.g., bakedproducts, comprising a protein hydrolysate obtained by the methods ofthe present invention. Such food products exhibit enhanced organolepticqualities, such as improvement in flavor, palatability, mouth feel,aroma and crust color.

In the present context, the term “baked products” includes any foodprepared from dough, either of a soft or a crisp character. Examples ofbaked products, whether of a white, light or dark type, which may beadvantageously produced by the present invention, are bread, inparticular white, whole-meal or rye bread, typically in the form ofloaves or rolls; French baguette-type breads; pita breads; tacos; cakes;pancakes; biscuits; crisp breads; and the like.

Such baked products are conventionally prepared from a dough whichcomprises flour and water, and which is typically leavened. The doughmay be leavened in various ways, such as by adding sodium bicarbonate orthe like, or by adding a leaven (fermenting dough), but the dough ispreferably leavened by adding a suitable yeast culture such as a cultureof Saccharomyces cerevisiae (baker's yeast). Any of the commerciallyavailable Saccharomyces cerevisiae strains may be employed.

Further, the dough used in the preparation of the baked products may befresh or frozen. The preparation of frozen dough is described by K. Kulpand K. Lorenz in “Frozen and Refrigerated Doughs and Batters”. A flavorimproving composition of the present invention is typically included inthe dough in an amount in the range of 0.01-5%, more preferably 0.1-3%.

In the methods of the present invention, a polypeptide of the presentinvention, an endopeptidase, a transglutaminase, a peptidoglutaminase,one or more specific and/or unspecific acting endo- and/or exo-peptidaseenzymes, and/or one or more enzymes specified above may be added, eitherseparately or concurrently, to the mixture from which the dough is madeor to any ingredient, e.g., flour, from which the dough is to be made.

The present invention further relates to a pre-mix, e.g., in the form ofa flour composition, for dough and/or baked products made from dough,wherein the pre-mix comprises a polypeptide or a flavor-improvingcomposition of the invention and a carrier or baking ingredient, andoptionally one or more other enzymes specified above.

In another embodiment, the pre-mix comprises a hydrolysate obtained bythe methods of the invention.

The pre-mix may be prepared by mixing the relevant enzymes with asuitable carrier such as flour, starch, a sugar or a salt. The pre-mixmay contain other dough-improving and/or bread-improving additives.

In the present context, the term “pre-mix” is a mixture of bakingagents, normally including flour, which has been prepared to permitstorage under designated conditions and provide convenience in handlingduring dough preparation processes. Such a pre-mix may be ofadvantageous use in industrial and commercial bread-baking plants andfacilities, as well as in retail bakeries.

The present invention also relates to the use of a hydrolysate producedby the methods of the invention as an additive to food products, such asbaked foods, to enhance organoleptic qualities, such as flavor,palatability and aroma.

The hydrolysates enriched in free glutamic acid and/or peptide boundglutamic acid residues obtained by the methods of the present inventionmay be used in various industrial applications, in particular, wherethere is a need for the incorporation of functional proteins.

For example, the present invention also relates to food productscomprising a hydrolysate enriched in free glutamic acid and/or peptidebound glutamic acid residues obtained by the method of the invention andto animal feed additives comprising a hydrolysate enriched in freeglutamic acid and/or peptide bound glutamic acid residues obtained bythe methods of the present invention.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Purification of FLAVOURZYME™ DipeptidylAminopeptidase I

Dipeptidyl aminopeptidase I was purified from a FLAVOURZYME™ broth (NovoNordisk A/S, Bagsvaerd, Denmark). The FLAVOURZYME™ broth was produced bycultivation of Aspergillus oryzae strain 1568 (ATCC 20386) in a mediumcomprised of carbon and nitrogen sources and trace metals. First, thebroth (20 ml containing 720 mg of protein) was diluted with 180 ml of 20mM sodium phosphate pH 7.0 buffer and filtered using Nalgene Filterwareequipped with a 0.45 μm filter. The filtered solution was loaded onto a24×130 mm column containing 31 ml of Q-Sepharose, Big Beads (PharnaciaBiotech AB, Uppsala, Sweden) pre-equilibrated with 20 mM sodiumphosphate pH 7.0 buffer. The dipeptidyl aminopeptidase I was elutedusing 20 mM sodium acetate buffer, pH 3.5. Dipeptidyl aminopeptidase Iactivity was monitored at 405 nm using 1 mg/ml Ala-Pro-p-nitroanilide assubstrate in 50 mM sodium phosphate pH 7.5 buffer. The resultantsolution containing dipeptidyl aminopeptidase I activity wasconcentrated to 38 ml by ultrafiltration with a PM1O membrane (Amicon,New Bedford, Mass.) and then the pH was adjusted to pH 7.0 using a 20 mMNa₂HPO₄ solution.

The resultant solution was loaded onto a 24×130 mm column containing 31ml of Q-Sepharose Big Beads (Pharnacia Biotech AB, Uppsala, Sweden)pre-equilibrated with 20 mM sodium phosphate pH 7.0 buffer. Thedipeptidyl aminopeptidase I was eluted with a 0 to 0.3 M NaCl gradientin 20 mM sodium phosphate pH 7.0 buffer. Fractions were monitored fordipeptidyl aminopeptidase I activity as described above. The fractionscontaining dipeptidyl aminopeptidase I activity were collected, pooled,desalted and concentrated using ultrafiltration against 20 mM sodiumphosphate pH 7.0 buffer.

The resultant solution was loaded onto a MonoQ 16/10 (20 ml) pre-packedcolumn (Pharmacia Biotech AB, Uppsala, Sweden) pre-equilibrated with 20mM sodium phosphate pH 7.0 buffer. The dipeptidyl aminopeptidase I waseluted with a 0 to 0.27 M NaCl gradient in 20 mM sodium phosphate pH 7.0buffer. Fractions were monitored for dipeptidyl aminopeptidase Iactivity as described above. The fractions between 0.200 and 0.212 MNaCl were collected, pooled, and rebuffered with 1.7 M (NH₄)₂SO₄/20 mMsodium phosphate pH 7.0 buffer using ultrafiltration as described above.

The resultant solution was loaded onto a 7×50 mm column containingPhenyl Superose resin (Pharmacia Biotech AB, Uppsala, Sweden)pre-equilibrated with 1.7 M (NH₄)₂SO₄/20 mM sodium phosphate pH 7.0buffer solution. The dipeptidyl aminopeptidase I was eluted with areverse 1.7 to 0 M (NH₄)₂SO₄ gradient in 20 MM sodium phosphate pH 7.0buffer. Fractions were monitored for dipeptidyl aminopeptidase Iactivity as described above. Two fractions possessing highest activitytoward Ala-Pro-p-nitroanilide were found to be at least 95% homogeneousbased on SDS-PAGE analysis. The major band had a molecular weight ofapproximately 95 kDa (range of 93-96 kDa).

Example 2 Protein Sequencing and Amino Acid Analysis Methods

N-terminal sequencing of the partially purified dipeptidylaminopeptidase I described in Example 1 and a digested fragment of thedipeptidyl aminopeptidase I was performed on an Applied Biosystems 476AProtein Sequencer (Perkin Elmer/Applied Biosystems Division, FosterCity, Calif.) with on-line HPLC and liquid phase trifluoroacetic acid(TFA) delivery. Samples of the purified dipeptidyl aminopeptidase I weretransblotted onto Novex PVDF membranes (Novex, San Diego, Calif.) fromSDS-PAGE gels and sequenced from a blott cartridge using sequencingreagents (Perkin Elmer/Applied Biosystems Division, Foster City,Calif.). Detection of phenylthiohydantoin-amino acids was accomplishedby on-line HPLC using Buffer A containing 3.5% tetrahydrofuran in waterwith 15 ml of the Premix concentrate (Perkin Elmer/Applied BiosystemsDivision, Foster City, Calif.) containing acetic acid, sodium acetate,and sodium hexanesulfonate and Buffer B containing acetonitrile. Datawas collected and analyzed on a Macintosh IIsi using Applied Biosystems610 Data Analysis software.

The partially purified dipeptidyl aminopeptidase I (MonoQ peak ofExample 1) was also subjected to in-gel digestion with trypsin togenerate peptide fragments of the enzyme for protein sequencing. Thetrypsin-digested dipeptidyl aminopeptidase I was separated by SDS-PAGEelectrophoresis using 8-16% Novex Tris-glycine gels (Novex, San Diego,Calif.). Eight Coommassie blue stained gel pieces corresponding to amolecular weight of 95 kDa were excised and washed extensively with 200mM NH₄HCO₃ in 50% acetonitrile. The gel pieces were subsequently reducedin 1 mM dithiothreitol (DTT) at 37° C. for 20 minutes and alkylated withan equal volume of 100 mM iodoacetic acid at room temperature for 20minutes in the dark. Gel pieces were washed repeatedly with 200 mMNH₄HCO₃ in 50% acetonitrile. Supernatants were removed and gel pieceswere dried on a Speed-Vac (Savant Instruments, Farmingdale, N.Y.). Thegel pieces were rehydrated in a solution containing 0.033 mg ofsequencing grade modified porcine trypsin (Promega, Madison, Wis.) perml of 135 mM NH₄HCO₃. The solution was prepared by diluting 1 part of0.1 mg of the trypsin per ml of trypsin resuspension buffer (Promega,Madison, Wis.) into 2 parts of 200 mM NH₄HCO₃. The gel pieces wereincubated at 37° C. for 20 hours. The peptide fragments were extractedfrom the gel in repetitive washes of 0.1% TFA in 60% acetonitrile for 1hour each. The extracted peptides were dried and reconstituted in 0.05%TFA in 25% acetonitrile and then further diluted into 0.05% TFA. Thepeptide fragments were filtered using a Micropure 0.45 μm filter unit(Amicon, Inc., Beverly, Mass.). The peptide fragments were thenseparated by reverse-phase HPLC using a Hewlett-Packard 1090L HPLCequipped with a 2.1×250 mm Vydac C 18-RP column (5 micron). A stepgradient was used with 0.1% TFA in 80% acetonitrile as the eluant. Thepeptide samples were hand collected and then subjected to N-terminalsequencing.

The partially purified dipeptidyl aminopeptidase I was also subjected tocyanogen bromide to generate peptide fragments of the enzyme forsequencing. The dipeptidyl aminopeptidase I was digested with cyanogenbromide by reconstituting a dried sample of the partially purified n 70%formic acid with a few crystals of cyanogen bromide and incubating for18 hours at room temperature in the dark. The peptide fragments wereseparated by SDS-PAGE electrophoresis using 10-20% Novex Tricine gels(Novex, San Diego, Calif.) and sequenced as described above.

N-terminal sequencing of the dipeptidyl aminopeptidase I revealed thatthe N-terminus was apparently blocked. A weak sequence was obtained asfollows where amino acid residues in parentheses are not 100% certainand residues marked with an X could not be determined:

Peptide 1: XEGSKRLTFXETVVKQAIT(P) (SEQ ID NO:3)

The cyanogen bromide-degraded fragments had the following amino acidsequences where amino acid residues underlined matched 100% with adipeptidyl aminopeptidase I of Saccharomyces cerevisiae (Anna-Arriolaand Herskowitz, 1994, Yeast 10: 801-810; Galisson and Dujon, 1996, Yeast12: 877-885):

Peptide 2: QRLPPGFSPDKKYPILFTPYGG (SEQ ID NO:4)

Peptide 3: KYIGPIK (SEQ ID NO:5)

Peptide 4: GEGSKRL (SEQ ID NO:6)

In-gel digestion with trypsin produced the following peptides whereamino acid residues underlined matched 100% with the deduced amino acidsequence of the Aspergillus oryzae dipeptidyl aminopeptidase I nucleicacid sequence described in Example 7:

Peptide 5: XPILFTPY (SEQ ID NO:7)

Peptide 6: XVPLMPDQ(Q)GDIOYAQ (SEQ ID NO:8)

Example 3 Genomic DNA Extraction

Aspergillus oryzae 1568 was grown in 25 ml of 0.5% yeast extract-2%glucose (YEG) medium for 24 hours at 37° C. and 250 rpm. Mycelia werethen collected by filtration through Miracloth (Calbiochem, La Jolla,Calif.) and washed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer.Excess buffer was drained from the mycelia preparation which wassubsequently frozen in liquid nitrogen. The frozen mycelia preparationwas ground to a fine powder in an electric coffee grinder, and thepowder was added to a disposable plastic centrifuge tube containing 20ml of TE buffer and 5 ml of 20% w/v sodium dodecylsulfate (SDS). Themixture was gently inverted several times to ensure mixing, andextracted twice with an equal volume of phenol:chloroform:isoamylalcohol (25:24:1 v/v/v). Sodium acetate (3 M solution) was added to theextracted sample to a final concentration of 0.3 M followed by 2.5volumes of ice cold ethanol to precipitate the DNA. The tube wascentrifuged at 15,000×g for 30 minutes to pellet the DNA. The DNA pelletwas allowed to air-dry for 30 minutes before resuspension in 0.5 ml ofTE buffer. DNase-free ribonuclease A was added to the resuspended DNApellet to a concentration of 100 μg/ml and the mixture was thenincubated at 37° C. for 30 minutes Proteinase K (200 μg/ml) was addedand the tube was incubated an additional one hour at 37° C. Finally, thesample was extracted twice with phenol:chloroform:isoamyl alcohol andthe DNA precipitated with ethanol. The precipitated DNA was washed with70% ethanol, dried under vacuum, resuspended in TE buffer, and stored at4° C.

Example 4 PCR Amplification of Aspergillus Oryzae 1568 DipeptidylAminopeptidase I

The forward degenerate oligonucleotide primer was designed to thepeptide sequence DW(I/V)YEEE, a conserved motif found in most publisheddipeptidyl aminopeptidase I protein sequences. The reverse degenerateoligonucleotide primer was designed to a partial peptide, PPGFSDKKYP, ofpeptide 2 (SEQ ID NO:4) as described in Example 2. The degenerateoligonucleotide primers shown below were synthesized with an AppliedBiosystems Model 394 DNA/RNA Synthesizer, according to themanufacturer's instructions, for use to PCR amplify dipeptidylaminopeptidase I gene fragments from Aspergillus oryzae 1568 genomicDNA:

Forward primer: 5′-GAYTGGITITAYGARGARGAR-3′ (SEQ ID NO:9) Reverseprimer: 5′-GGRTAYTTYTTRTCIGGISWRAAICCIGGIGG-3′ (SEQ ID NO:10)

(R=A or G, Y=C or T, S=G or C, W=A or T, I=Inosine)

Amplification reactions (100 μl) were prepared using approximately 1 μgof genomic DNA isolated from an Aspergillus oryzae 1568 as described inExample 3 as the template. Each reaction contained the followingcomponents: 1 μg genomic DNA, 40 pmol forward primer, 40 pmol reverseprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1×Taq polymerasebuffer (Perkin-Elmer Corp., Branchburg, N.J.), and 2.5 Units of Taqpolymerase (Perkin-Elmer Corp., Branchburg, N.J.). The reactions wereincubated in a Perkin-Elmer Model 480 Thermal Cycler programmed asfollows: Cycle 1 at 94° C. for 2 minutes, 45° C. for one minutes, and72° C. for one minute; and cycles 2-30 each at 94° C. for one minute,45° C. for one minute, and 72° C. for one minute. The reaction productswere isolated on a 1% agarose gel (Eastman Kodak, Rochester, N.Y.). Anapproximately 1.0 kb product band was excised from the gel and purifiedusing GenElute spin columns (Supelco, Bellefonte, Pa.) according to themanufacturer's instructions. The purified PCR product was subsequentlycloned into a pCRII vector (Invitrogen, San Diego, Calif.) and the DNAsequence was determined using lac forward and reverse primers (NewEngland BioLabs, Beverly, Mass.).

A dipeptidyl aminopeptidase I gene segment consisting of approximately321 codons (963bp) was amplified from Aspergillus oryzae 1568 with thedipeptidyl aminopeptidase I PCR primers described above. DNA sequenceanalysis shows that the amplified gene segment encoded a portion of thecorresponding Aspergillus oryzae 1568 dipeptidyl aminopeptidase I gene.The dipeptidyl aminopeptidase I gene segment was used to probe anAspergillus oryzae 1568 genomic DNA library.

Example 5 Construction of DNA Libraries

A genomic DNA library was constructed in the bacteriophage cloningvector λZipLox (Life Technologies, Gaithersburg, Md.). First, totalcellular DNA was partially digested with Tsp509I and size-fractionatedon 1% agarose gels. DNA fragments migrating in the size range 3-7 kbwere excised and eluted from the gel using Prep-a-Gene reagents (BioRadLaboratories, Hercules, Calif.). The eluted DNA fragments were ligatedwith EcoRI-cleaved and dephosphorylated λZipLox vector arms (LifeTechnologies, Gaithersburg, Md.), and the ligation mixtures werepackaged using commercial packaging extracts (Stratagene, La Jolla,Calif.). The packaged DNA libraries were plated and amplified inEscherichia coli Y1090ZL cells (Life Technologies, Gaithersburg, Md.).The unamplified genomic DNA library contained 3.1×10⁶ pfu/ml (backgroundtiters with no DNA were 2.0×10⁴ pfu/ml).

Example 6 Identification of Dipeptidyl Aminopeptidase I Clones

Approximately 10,000 plaques from the library described in Example 5were screened by plaque-hybridization using the dipeptidylaminopeptidase I PCR fragment from Aspergillus oryzae 1568 as the probe.The DNA was cross-linked onto membranes (Hybond N+, Amersham, ArlingtonHeights, Ill.) using a UV Stratalinker (Stratagene, La Jolla, Calif.).The membranes were soaked for three hours at 45° C. in a hybridizationsolution containing 5×SSPE, 0.3% SDS, 50% formamide, and 10 μg/ml ofdenatured and sheared herring sperm DNA. The dipeptidyl aminopeptidase Igene fragment isolated from the Aspergillus oryzae 1568 genomic DNA asdescribed in Example 2 was radiolabeled using the Random Primed DNALabeling Kit (Boehringer Mannheim, Mannheim, Germany), denatured byadding NaOH to a final concentration of 0.1 M, and added to thehybridization solution at an activity of approximately 1×10⁶ cpm per mlof hybridization solution. The mixture was incubated overnight at 45° C.in a shaking water bath. Following incubation, the membranes were washedonce in 2×SSC with 0.2% SDS at 55° C. followed by two washes in 2×SSC atthe same temperature. The membranes were dried on blotting paper for 15minutes, wrapped in SaranWrap™, and exposed to X-ray film overnight at−70° C. with intensifying screens (Kodak, Rochester, N.Y.).

Based on the production of strong hybridization signals with the probe,three plaques, designated E. coli DH5α MWR52A, E. coli DH5α MWR52B, andE. coli DH5α MWR52C were chosen for further study. The three plaqueswere purified twice in E. coli Y1090ZL cells and the dipeptidylaminopeptidase I genes were subsequently excised from the λZipLox vectoras pZL1-derivatives (D'Alessio et al., 1992, Focus® 14:76) using in vivoexcision by infection of E. coli DH10BZL cells (Life Technologies,Gaithersburg, Md.). The three plasmid containing colonies wereinoculated into three ml of LB plus 50 μg/ml carbenicillin medium andgrown overnight at 37° C. Miniprep DNA was prepared from each of thesecultures using the Wizard 373 DNA Purification Kit (Promega, Madison,Wis.). The dipeptidyl aminopeptidase I encoding plasmid (pMWR52) wasconfirmed by DNA sequencing.

Example 7 DNA Sequence Analysis of Aspergillus oryzae 1568 DipeptidylAminopeptidase I Gene

DNA sequencing of the dipeptidyl aminopeptidase I gene contained onpMWR52 in E. coli DH5α MWR52 described in Example 6 was performed withan Applied Biosystems Model 377 Automated DNA Sequencer (AppliedBiosystems, Inc., Foster City, Calif.) on both strands using the primerwalking technique with dye-terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60). Oligonucleotide sequencingprimers were designed to complementary sequences in the dipeptidylaminopeptidase I gene and were synthesized on an Applied BiosystemsModel 394 DNA/RNA Synthesizer according to the manufacturer'sinstructions.

The nucleotide sequence of the gene encoding the Aspergillus oryzae 1568dipeptidyl aminopeptidase I is shown in FIG. 1 (SEQ ID NO:1). Sequenceanalysis of the cloned insert revealed an open reading frame of 2396nucleotides (excluding the stop codon) interrupted by an 83 bp intron.The G+C content of this open reading frame was 55.3%. The deduced aminoacid sequence encoded a protein of 771 amino acids (SEQ ID NO:2). Basedon the rules of van Heijne (van Heijne, 1984, Journal of MolecularBiology 173: 243-251), the first 16 amino acids likely comprise asecretory signal peptide which directs the nascent polypeptide into theendoplasmic reticulum (boxed in FIG. 1).

The amino acid sequences of the partial peptides derived from thepurified dipeptidyl aminopeptidase I as described in Example 2 areunderlined in FIG. 1 and are consistent with those found in the deducedamino acid sequence (SEQ ID NO:2) of the Aspergillus oryzae 1568dipeptidyl aminopeptidase I cDNA.

Using the Clustal alignment program (Higgins, 1989, CABIOS 5: 151-153)to compare the deduced amino acid sequence of the Aspergillus oryzae1568 dipeptidyl aminopeptidase I to that of a Saccharomyces cerevisiaedipeptidyl aminopeptidase I (Anna-Arriola and Herskowitz, 1994, Yeast10: 801-810) (SEQ ID NO:11), a 23.2% identity was observed.

Example 8 Construction of a Aspergillus oryzae 1568 DipeptidylAminopeptidase Fusarium Expression Vector

The coding region of Aspergillus oryzae dipeptidyl aminopeptidase I wasamplified and the resulting fragment was cloned into pDM181 forexpression in Fusarium. pDM181 provides the Fusarium trypsin (SP387)promoter and terminator, and the bar selectable marker gene.Specifically, the fragment was amplified by PCR using a sense primer(P1) designed to the first in-frame ATG and extending 13 bp downstreamand an antisense primer (P2) designed to a region of the transcriptionalstop codon and extending 10 bp downstream. To facilitate the cloning ofthe amplified fragment the sense and antisense primers contain a Swaland a PacI restriction site, respectively. The oligonucleotide primersshown below were synthesized using an ABI Model 394 DNA/RNA Synthesizer(Applied Biosystems, Inc., Foster City, Calif.) according to themanufacturer's instructions.

Swal

P1: 5′-GATTTAAATCACCATGAAGGTACGTCAATTCCACTG-3′ (SEQ ID NO:12)

PacI

P2: 5′-GTTAATTAATCTACTCCTCCAAGTCCTTCTTAGTCC-3′ (SEQ ID NO:13)

The 50 μl PCR solution (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂,0.01% w/v gelatin) contained approximately 200 ng of pMWR52 DNA, 200 μMeach of dATP, dCTP, dGTP, and dTTP, and 50 pmol of each PCR primerdescribed above. Five units of PWO polymerase (Boehringer Mannheim,Indianapolis, Ind.) were added and the reaction was incubated at 95° C.for 3 minutes and cooled to 80° C. The reaction was then cycled 30times, each cycle at 95° C. for 30 seconds, 57° C. for 1 minute, and 72°C. for 1 m Perkin-Elmer 9600 Thermal Cycler. Following the last cycle,the reaction incubated for 5 minutes at 72° C.

A predicted 2.4 kb fragment was isolated by digestion with Swal and PacIand was cloned into pDM181 (FIG. 2) digested with the same restrictionendonucleases to create pMWR54 (FIG. 3). To verify the fidelity of thecloned PCR fragment, the fragment was sequenced according to the methodof Hattori and Sakaki (1986, Analytical Biochemistry 152: 232-237) usingan automated Applied Biosystems Model 373A Sequencer (AppliedBiosystems, Inc., Foster City, Calif.) according to the manufacturer'sinstructions.

Sequencing of the cloned dipeptidyl aminopeptidase amplified insert ofpMWR54 confirmed that there were no differences in the sequencedescribed in SEQ ID NO:1.

Example 9 Transformation of Fusarium CC1-3 and Analysis of Transformants

Fusarium strain CC1-3, a highly branched morphological mutant ofFusarium strain A3/5 (ATCC 20334) (Wiebe et al., 1992, MycologicalResearch 96: 555-562; Wiebe et al., 1991, Mycological Research 95:1284-1288; Wiebe et al., 1991, Mycological Research 96: 555-562), wasgrown in a liquid medium containing Vogel's salts, (Vogel, 1964, Am.Nature 98: 435-446), 25 mM NaNO₃, and 1.5% glucose for 4 days at 28° C.and 150 rpm. Conidia were purified by filtration through 4 layers ofcheesecloth and finally through one layer of Miracloth. Conidialsuspensions were concentrated by centrifugation. Fifty ml of YPG mediumcomprised of 1% yeast extract, 2% bactopeptone, and 2% glucose wereinoculated with approximately 108 conidia, and incubated for 14 hours at24° C. and 150 rpm. Resulting hyphae were trapped on a sterile 0.4 mmfilter and washed successively with sterile distilled water and 1.0 MMgSO₄. The hyphae were resuspended in 10 ml of NOVOZYM 234™ solution(2-10 mg/ml in 1.0 M MgSO₄) and digested for 15-30 minutes at 34° C.with agitati 80 rpm. Undigested hyphal material was removed from theresulting protoplast suspension by successive filtration through 4layers of cheesecloth and through Miracloth. Twenty ml of 1 M sorbitolwere combined with the protoplast solution. After mixing, theprotoplasts were pelleted by centrifugation and washed successively byresuspension and centrifugation in 20 ml of 1 M sorbitol and in 20 ml ofSTC (0.8 M sorbitol, 0.05 M Tris pH 8.0, 0.05 M CaCl₂). The washedprotoplasts were resuspended in 4 parts STC and 1 part SPTC (0.8 Msorbitol, 40% PEG 4000, 0.05 M Tris pH 8.0, 0.05 M CaCl₂) at aconcentration of 5×10⁷/ml.

One hundred ml of protoplast suspension were added to 10 μg of pMWR54 inpolypropylene tubes (17×100 mm), mixed and incubated on ice for 30minutes. One ml of SPTC was mixed gently into the protoplast suspensionand incubation was continued at room temperature for 20 minutes. 12.5 mlof molten solution (cooled to 40° C.) consisting of 1X Vogel's salts, 25mM NaNO₃, 0.8 M sucrose and 1% low melting agarose (Sigma ChemicalCompany, St. Louis, Mo.) were mixed with the protoplasts and then platedonto an empty 100 mm Petri plate. Incubation was continued at roomtemperature for 10 to 14 days. After incubation at room temperature for24 hours, 12.5 ml of the identical medium plus 10 mg of BASTA™ (HoechstSchering, Rodovre, Denmark) per ml were overlayed onto the Petri plate.BASTA™ was extracted twice with phenol:chloroform:isoamyl alcohol(25:24:1), and once with chloroform:isoamyl alcohol (24:1) before use.

After two weeks, 21 transformants were apparent. A mycelial fragmentfrom the edge of each transformant was transferred to individual wellsof a 24 well plate containing Vogel's/BASTA™ medium. The medium wascomposed per liter of 25 g of sucrose, 25 g of Noble agar, 20 ml of 50XVogel's salts (Vogel, 1964, supra), 25 mM NaNO₃, and 10 g of BASTA™. Theplate was sealed in a plastic bag to maintain moisture and incubatedapproximately one week at room temperature.

Example 10 Expression of Aspergillus Oryzae 1568 DipeptidylAminopeptidase I in Fusarium

A mycelial fragment from each of the 21 Fusarium CC1-3 transformantsdescribed in Example 9 was inoculated into 20 ml of M400Da mediumcomposed per liter of 50 g of maltodextrin, 2.0 g of MgSO₄-7H₂0, 2.0 gof KH₂PO₄, 4.0 g of citric acid, 8.0 g of yeast extract, 2.0 g of urea,and 0.5 ml of trace metals solution and incubated for 5 days at 30° C.and 200 rpm. The medium was adjusted to pH 6.0 with 5 N NaOH. The tracemetals solution was compsoed per liter of 14.3 g of ZnSO₄-7H₂O, 2.5 g ofCuSO₄-5H₂0, 0.5 g of NiCl₂O, 13.8 g of FeSO₄-7H₂O, 8.5 g of MnSO₄-H₂O,and 3.0 g of citric acid. The untransformed host was also run as acontrol. One ml of each culture supernatant was harvested at 5 days andstored at 4° C. Dipeptidyl aminopeptidase I activity was determined bymixing 1 μl of the enzyme supernatant with 200 μl of a Ala-Pro-pNAsubstrate stock solution containing 2 mg of Ala-Pro-pNA per ml of 50 mMsodium phosphate pH 7.5 and monitoring the change in absorbance at 405nm and ambient temperature.

Culture supernatants from 19 of the 21 primary transformants of pMWR54were positive when assayed for dipeptidyl aminopeptidase I activity.

The Fusarium primary transformants #1 and #16 were cultivated in 125 mlshake flasks for 5 days at 30° C. in 25 ml of M400Da medium. The wholeculture broths from the Fusarium primary transformants were filteredusing a double layer of Miracloth. The filtrate was recovered and thenfrozen at −20° C.

Example 11 Purification of Recombinant Aspergillus oryzae 1568Dipeptidyl Aminopeptidase I Produced in Fusarium

The recombinant dipeptidyl aminopeptidase I was purified from theFusarium broth of primary transformant #1 described in Example 10. Thebroth (20 ml) was filtered through Nalgene filterware equipped with a0.45 micron filter (Nalgene, Rochester, N.Y.). The sample was diluted10-fold using 20 mM sodium phosphate pH 7.0 buffer and concentratedusing a ultrafiltration system (Amicon, Beverly, Mass.) utilizing a PM10ultrafiltration membrane. The conductivity of the sample was 2.5 mS.

The sample was then loaded onto a column (XK-26) containing 60 ml ofQ-Sepharose Big Beads, which had been pre-equilibrated with 400 ml of 20mM sodium phosphate pH 7.0 buffer. The column was washed until baselinewas reached. At a flow rate of 5 ml/min, the dipeptidyl aminopeptidase Iwas eluted with a linear gradient from 0-0.40 M NaCl in 20 mM sodiumphosphate buffer pH 7.0 buffer over 10 column volumes. The dipeptidylaminopeptidase I eluted at ˜0.24 M NaCl. SDS-PAGE was performed on thefractions active Ala-Pro-pNA. The substrate was prepared by dissolving 2mg of Ala-Pro-pNA (Bachem, Torrance, Calif.) in 20 μl of DMSO. Then, 980μl of 50 mM sodium phosphate pH 7.5 buffer was added. In a 96 wellmicrotiter plate, 100 μl of the substrate solution was added to 100 μlof Aspergillus oryzae dipeptidyl aminopeptidase I (100 fold diluted in50 mM sodium phosphate buffer pH 7.5) and the increase in absorbance at405 nm was measured for 4 minutes using a SpectroMax 340 plate reader(Molecular Devices, Sunnyvale, Calif.). The homogeneous fractions werethen pooled.

Example 12 Characterization of Recombinant Aspergillus oryzae 1568Dipeptidyl Aminopeptidase I

The purified dipeptidyl aminopeptidase I described in Example 11 wascharacterized with respect to pH optimum, temperature stability,substrate specificity, and kinetic parameters.

The pH optimum was determined using Ala-Pro-pNA (HCI salt) as substratein the universal buffer composed of 0.125 M citric acid, 0.125 M monobasic sodium phosphate, and 0.125 M boric acid pH was adjusted to4.35-9.83 with 10 N NaOH, in 0.5 pH increments. The Ala-Pro-pNAsubstrate was prepared by dissolving 100 mg of Ala-Pro-pNA in 1 ml ofDMSO and adding 20 μl of the Ala-Pro-pNA-DMSO solution to 980 μl of theuniversal buffer at the various pH values at ambient temperature. Theassay was initiated by adding a 10 μl aliquot of the dipeptidylaminopeptidase I diluted 20-fold in water to 200 μl of 2 mg/mlAla-Pro-pNA at the various pH values. The absorbance at 405 nm wasmonitored for 5 minutes. Autohydrolysis of the substrate as a controlwas determined by adding 10 μl of water to 200 μl of 2 mg/ml Ala-Pro-pNAat the various pH values.

The results shown in Table I below demonstrated that the dipeptidylaminopeptidase I possessed activity toward Ala-Pro-pNA as substrate overthe measured pH range 4.35 to 9.83 with optimal activity at pH ˜8.7.Autohydrolysis of the substrate was observed at pH values greater than7.

TABLE 1 Average Average Avg. Relative pH Activty BackgroundActivity-Background Activty 4.35  8.75 mOD/min  0 mOD/min  8.75 mOD/min0.023 4.87  32.5  0  32.5 0.088 5.36  75.75  0  75.75 0.20 5.86 113.8  0113.8 0.307 6.38 135.48  0 135.48 0.365 6.85 168.45  2 166.45 0.45 7.2188.86  2 186.86 0.503 7.51 230.97  3 227.97 0.615 7.97 308.52  5.8302.72 0.817 8.71 383.15 12.5 370.65 1 9.32 247.68 29.8 217.88 0.5889.83 171 74  97 0.261

The temperature stability of the dipeptidyl aminopeptidase I wasdetermined using the following protocol: 490 μl of 50 mM sodiumphosphate buffer pH 7.5 was preincubated at 37°, 45°, 55°, 60°, 65°,70°, and 75° C. for 30 minutes in a 1.7 ml Eppendorf tube. Then 10 μl ofpurified dipeptidyl aminopeptidase I was added and the sample was thenincubated for an additional 20 minutes. The samples were then placed onice. Once the incubations were completed for all the temperatures, thesamples were then assayed for activity using Ala-Pro-pNA as substrate.

The assay was performed by mixing 30 μl of the incubation mixtures forthe various temperatures with 200 μl of 2 mg/ml Ala-Pro-pNA in 50 mMsodium phosphate pH 7.5 buffer) at ambient temperature. The absorbanceat 405 nm was monitored for 5 minutes.

The results shown in Table 2 demonstrated that the dipeptidylaminopeptidase I retained 90% of its activity after a 20 minuteincubation at 65° C., pH 7.5.

TABLE 2 Temperature (° C.) Percent activity relative to 37° C. 37 100 45103 55 103 65 92.5 70 37.7 75 0.7

The relative activity of various mono-, di-, and tri-peptidepara-nitroanilide substrates were assayed relative to Ala-Pro-pNA withthe purified dipeptidyl aminopeptidase I diluted 100-fold in 50 mMsodium phosphate pH 7.5 buffer. Each substrate was dissolved in DMSO toa concentration of 100 mg/ml and then diluted 50 fold in 50 mM sodiumphosphate pH 7.5 buffer to 2 mg/ml. The assay was performed by mixing100 μl of the substrate solution with 100 μl of the dipeptidylaminopeptidase I solution and monitoring the change in absorbance at 405nm and ambient temperature.

The results shown in Table 3 demonstrated that the dipeptidylaminopeptidase I preferably hydrolyzed Xaa-Pro-pNA and Xaa-Ala-pNAsubstrates where Xaa corresponds to any natural amino acid.

TABLE 3 Percent Activity Relative to Substrate: Activity (mOD/min)Ala-Pro-pNA Gly-Arg-pNA 0 0 Gly-Pro-pNA 125 51.25 Arg-Pro-pNA 103 42.17Val-Ala-pNA 49.5 20.3 Gly-Glu-pNA <2 0 Ala-Pro-pNA 244 100 Ala-Ala-pNA30.2 12.4 Asp-Pro-pNA 70.95 29 Leu-pNA 0 0 Ala-pNA 0 0 Ala-Ala-Pro-pNA10.6 4.3

The kinetic parameters for various dipeptidyl aminopeptidase Isubstrates was determined using the following protocol. The substratesincluded Ala-Pro-pNA, Asp-Pro-pNA, and Ala-Ala-pNA. Purified dipeptidylaminopeptidase I with an A₂₈₀ of 1.521 was diluted 25-fold forAla-Pro-pNA, 20-fold for Asp-Pro-pNA, and 10-fold for Ala-Ala-pNA inwater. Each substrate was dissolved in DMSO to a concentration of 100mg/ml and then diluted 50 fold in 50 mM sodium phosphate pH 7.5 bufferto 2 mg/ml. In a 96 well microtiter plate, 10 μl of purified dipeptidylaminopeptidase I was incubated with each substrate as follows except the200 μl substrate assay was not performed with the Asp-Pro-pNA substrate,and the absorbance at 405 nm was measured for 3 minutes:

1. 200 μl of 2 mg/ml substrate+0 μl of 50 mM sodium phosphate buffer pH7.5

2. 100 μl of 2 mg/ml substrate+100 μl of 50 mM sodium phosphate bufferpH 7.5

3. 50 μl of 2 mg/ml substrate+150 μl of 50 mM sodium phosphate buffer pH7.5

4. 25 μl of 2 mg/ml substrate+175 μl of 50 mM sodium phosphate buffer pH7.5

5. 10 μl of 2 mg/ml substrate+190 μl of 50 mM sodium phosphate buffer pH7.5

6. 5 μl of 2 mg/ml substrate+195 μl of 50 mM sodium phosphate buffer pH7.5

A Lineweaver-Burke plot was constructed to determine the K_(m) and thek_(cat) for each substrate, using an average molecular weight of 97 kDafor the differentially glycosylated forms.

For Ala-Pro-pNA, the K_(m) and k_(cat) were determined to be 0.140 mMand 576.3 min⁻¹, respectively.

For Asp-Pro-pNA, the K_(m) and k_(cat) were determined to be 0.632 mMand 244.3 min⁻¹, respectively.

For Ala-Ala-pNA, the K_(m) and k_(cat) were determined to be 1.08 mM and106.5 min⁻¹, respectively.

Example 13 Purification of Aspergillus oryzae 1568 Aminopeptidase I

A 50 ml volume of FLAVOURZYME™ preparation described in Example 1 wascentrifuged at 10,000 rpm for 10 minutes. The supernatant was filteredwith a 0.2 μm Nalgene filter and the filtrate was then diluted to 350 mlwith 20 mM sodium phosphate pH 7.5 buffer. The diluted filtrate wasconcentrated with an Amicon ultrafiltration cell equipped with a PM10membrane. More 20 mM phosphate pH 7.5 buffer was added and concentrated3 more times to adjust the sample to the proper pH and conductivity.

The enzyme solution was then loaded onto a XK−26 column containingQ-Sepharose Big Beads pre-equilibrated with 20 mM phosphate buffer pH7.5 buffer. A gradient was run from 0 to 300 mM NaCl, and then washedwith 350 ml of 300 mM NaCl. Fractions were assayed for Leu-pNA activityaccording to the following protocol. The substrate was prepared bydissolving 2 mg of Leu-pNA (Sigma Chemical Co., St. Louis, Mo.) in 20 μlof DMSO.

Then, 980 μl of 50 mM sodium phosphate pH 7.5 buffer was added. In a 96well microtiter plate, 100 μl of the substrate solution was added to 100μl of Aspergillus oryzae aminopeptidase I (100 fold diluted in 50 mMsodium phosphate pH 7.5 buffer) and the increase in absorbance at 405 nmwas measured for 4 minutes using a SpectroMax 340 plate reader. The mostactive fractions were pooled and concentrated as above using PM10ultrafiltration.

The concentrated sample was diluted to 250 ml with 20 mM phosphate pH7.5 buffer and loaded onto a Mono-Q 16/10 column (Pharmacia Biotech AB,Uppsala, Sweden). A gradient was run from 0 to 400 mM NaCl. Fractionswere assayed for Leu-pNA activity as above. The most active and purefractions (by SDS-PAGE) were pooled and concentrated as above usingusing PM10 ultrafiltration.

The sample was diluted in 50 mM phosphate pH 7.0 buffer with 1.7 Mammonium sulfate and concentrated as above. This step was repeated threetimes. The sample was loaded onto Phenyl Sepharose column (PharmaciaBiotech AB, Uppsala, Sweden) pre-equilibrated with 50 mM sodiumphosphate pH 7.0 buffer containing 1.7 M ammonium sulfate. A gradientwas run from 1.7 M to 0 M ammonium sulfate, and then washed with 50 mMsodium phosphate pH 7.0 buffer. Fractions were assayed for Leu-pNAactivity and purity was checked by SDS-PAGE. Two small bands near 70 kDawere still present.

A 1 ml volume of the most pure fraction was concentrated with a Microcon10 microconcentrator (Amicon, New Bedford, Mass.). A 100 μl volume ofthe concentrate was loaded onto a Superose 12 column (Pharmacia BiotechAB, Uppsala, Sweden) pre-equilibrated with 20 mM phosphate buffer pH 7.0buffer, and eluted in 30 ml of the same buffer. Only one peak wasobserved. SDS-PAGE analysis revealed two small bands near 70 kDa. Thesebands were probably an aggregation of the dipeptidyl aminopeptidase, andno further purification was necessary.

All active fractions from the Phenyl Sepharose column were pooled,concentrated, and desalted using PM10 ultrafiltration and 20 mMphosphate pH 7.0 buffer.

Example 14 Synergism between Aspergillus oryzae 1568 aminopeptidase Iand Aspergillus oryzae 1568 Dipeptidyl Aminopeptidase I

The substrate was prepared by dissolving 2 mg of Ala-Phe-Pro-pNA(Bachem, Torrance, Calif.) in 20 μl of DMSO. Then, 980 μl of 50 mMsodium phosphate buffer pH 7.5 and 100 μl of ethanol were added. In a 96well microtiter plate, 100 l of the Ala-Phe-Pro-pNA solution was addedto 100 μl of 100-fold diluted aminopeptidase I (Example 12); 100 μl ofthe Ala-Phe-Pro-pNA solution was added to 100 μl of 100-fold diluteddipeptidyl aminopeptidase I (Example 11); and 100 μl of theAla-Phe-Pro-pNA solution was added to a mixture of 50 μl of 100-folddiluted aminopeptidase I and 50 μl of 100-fold diluted dipeptidylaminopeptidase I. The absorbance at 405 nm was measured for 10 minutes.

The purified aminopeptidase I was determined to have an activity of26.822 LAPUs/ml for Leu-pNA using an extinction coefficient of 10,000for para-nitroaniline at 405 nm. LAPU is defined as the leucineaminopeptidase activity which is determined as described in AF 298/1-GB(available on request from Novo Nordisk A/S, Denmark). The purifieddipeptidyl aminopeptidase I was determined to have an activity of 11.54DPAPU/ml for Ala-Pro-pNA, also using an extinction coefficient of 10000for paranitroaniline. At a 100-fold dilution, aminopeptidase I exhibitedless than a 2 mOD/min velocity on Ala-Pro-pNA and dipeptidylaminopeptidase I exhibited less than a 2 mOD/min velocity on Leu-pNA.One hundred-fold diluted aminopeptidase I exhibited no activity onAla-Phe-Pro-pNA over 10 minutes. One hundred-fold diluted dipeptidylaminopeptidase I exhibited no activity on Ala-Phe-Pro-pNA over 10minutes. When a mixture of 100-fold diluted dipeptidyl aminopeptidase Iand 100-fold diluted aminopeptidase I were assayed with Ala-Phe-Pro-pNA,a velocity of 122 mOD/min was observed over 10 minutes.

Example 15 Preparation of Protein Hydrolysates with Aspergillus oryzae1568 Dipeptidyl Aminopeptidase I

The purified dipeptidyl aminopeptidase I described in Example 11 wastested in degree of hydrolysis assays using geletin, soy, gluten, andcasein as substrates according to the following procedure.

The degree of hydrolysis (DH) assays were performed at 50° C. for 18hours as a mini-hydrolysis on a 10 ml scale using gelatin, soy bean mealtablets, wheat gluten, and sodium-caseinate at a 2% concentrationadjusted to pH 7, if necessary, with no pH adjustment during hydrolysis.The hydrolyses were inactivated at 85° C. for 3 minutes in a waterbath.The enzymes used were FLAVOURZYME™ with ALCALASE™ 2.4L (Novo NordiskA/S, Bagsvrd, Denmark) and dipeptidyl aminopeptidase I with FLAVOURZYME™and ALCALASE™ 2.4L. FLAVOURZYME™, ALCALASE™, and dipeptidylaminopeptidase I were dosed at 3 mg, 3 mg, 0.125 mg, respectively, per200 mg of protein per ml.

The DH, as defined by Adler-Nissen (1986, Enzymic Hydrolysis of FoodProteins, Elsevier Applied Science Publishers), was determined byreaction of the supernatant with OPA (ortho-phtaldialdehyde, SigmaChemical Co., St. Louis, Mo.). For the OPA reagent, 160 mg of OPA wasdissolved in 4 ml of ethanol and transferred to a 200 ml volumetricflask containing a solution of 7.62 g of disodium tetraboratedecahydrate, 200 mg of sodium dodecylsulphate, and 176 mg ofdithiothreitol and the flask was filled to 200 ml with water.

A volume of 25 μl of suitably diluted supernatant was mixed with 200 μlof OPA reagent in a microtiter plate well and allowed to react forexactly 2 minutes at 25° C. The absorbance at 340 nm was measured in amicrotiter plate reader and compared to the absorbance of a 95 mML-serine standard solution after subtraction of the blank value (waterreacted with OPA-reagent). To determine the true DH, the serineequivalents measured in the supernatants were corrected with the factorssuggested by Adler-Nissen for the trinitrobenzenesulfonic acid method(Adler-Nissen, 1979, Agricultural and Food Chemistry 17: 1256) whichgave the same response as the described OPA method. The degree ofhydrolysis was calculated on basis of the total amount of protein in thehydrolysis mixture (not on basis of soluble protein).

The results showed that dipeptidyl aminopeptidase increased the degreeof hydrolysis 5% above the samples with FLAVOURZYME™ and ALCALASE™ alonefor all the proteins tested.

Example 16 Increased Protein Solubility and Release of Glutamate byDeamidation

Wheat gluten (WG) was obtained from Cargill (JOB 5141) and deamidatedwheat gluten (DWG) was obtained from StaPro Consultancy B.V., Lemdijk32, 9422 TH Smilde, NL. Suspensions of 8% protein were made by mixing 11g of gluten with 89 g of water. The pH was adjusted to 6.5 with NaOH.Glutamate/aspartate specific protease (SP446), obtainable as describedin WO 91/13554, or lysine/arginine specific protease (SP387) obtainableas described in WO 89/06270, was added to the suspensions. The dosagewas 0.01 AU/g protein for SP446 and 0.006 AU/g protein for SP387.FLAVOURZYME™ (an non-specifically acting protease preparation availablefrom Novo Nordisk A/S, Bagsvaerd, Denmark, containing endo- andexo-peptidase activities, and obtained by fermentation of Aspergillusoryzae) was added to some of the hydrolysates at a dosage of 20 LAPU/gprotein. One LAPU (Leucine Amino Peptidase Unit) is the amount of enzymewhich decomposes 1 micromole of L-leucine-p-nitroanilide per minuteunder the following conditions: 26 mM L-leucine-p-nitroanilide in 0.1 MTris pH 8.0 buffer at 40° C. for 10 minutes. Upon hydrolysis,p-nitroanilide is liberated turning the solution yellow which ismonitored 405 nm.

The hydrolyses were carried out at 50° C. without further pH adjustmentfor 18 hours. The enzymes were inactivated by heating at 85° C. for 15minutes. The pH was adjusted to 5 and the hydrolysates were centrifuged.The content of protein and free glutamate in the supernatant wasdetermined.

The protein content was determined by Kjeldahl analysis, using aKjeldahl factor of 6.25.

The content of free glutamate was determined by use of a glutamatedetermination kit according to the manufacturer's instructions(Boehringer-Mannheim, Indianapolis, Ind.). The method was adapted foruse in microtiter plates.

When comparing wheat gluten (WG) to deamidated wheat gluten (DWG), theresults as shown in Table 4 demonstrated that deamidation increased thesusceptibility of the gluten to specific proteases, such that moreprotein became soluble. By addition of FLAVOURZYME™ with a specificprotease, the release of glutamate was doubled due to deamidation.

TABLE 4 Protein Solubility Glutamate Content % mg/l Hydrolysate WG DWGWG DWG SP446 18 54 0 0 SP387 35 44 0 0 SP446 + 34 87 1000 2000  FLAVOURZYME ™

Example 17 Enzymatic Deamidation and Release of Glutamate

Peptidoglutaminase II was produced by growing Bacillus circulans strainATCC 21590 in shake flasks (400 ml) containing 200 ml of a mediumcomposed of 1% polypeptone, 0.5% lactose, 0.025% MgSO₄-7H₂O, 0.005%FeSO₄-7H₂O, 0.025% KH₂PO₄, and 17% Na₂HPO₄-12H₂O (pH adjusted to 7.2),at 30° C. for 20 hours with mixing at 270 rpm. The cells were harvestedby centrifugation at 4000 rpm in 1 litre flasks. The cells were thenfrozen.

The purification of peptidoglutaminase II from Bacillus circulans wasperformed at room temperature. The frozen Bacillus circulans cells werethawed and suspended in Lysis buffer (50 mM Tris/HCI; 25% (w/v) sucrose;1 mM EDTA, pH 8.0) until a homogeneous suspension was obtained—100 g wetcells per liter of Lysis buffer. Lysozyme (10 mg/ml) and DNAse I (SigmaDN-25, 10 mg/ml) were dissolved in Lysis buffer. Then 100 ml of lysozymesolution, 10 ml of 1.0 M MgCl₂, and 1 ml of DNAse I solution were addedper litre of cell suspension. The enzymes were allowed to act for 1hour.

The suspension was filtered through a Seitz depth filter plate and thefiltrate was transferred to a 10 mM KH₂PO₄/NaOH, pH 8.0 (Buffer A) on aSephadex G25 column (Pharmacia). The enzyme solution was applied to aSOURCE Q column (Pharmacia) equilibrated in Buffer A and eluted with alinear NaCl gradient (0→500 mM) in Buffer A. Fractions from the columnwere analysed for Peptidoglutaminase II activity as described below andfractions with activity were pooled. The absorbance of the pooledfractions at 280 nm was 1.78, thus the protein content was estimated to1.8 mg/ml.

The purity of the protein in the peptidoglutaminase II pool wasapproximately 25% as judged from a SDS-PAGE gel. Thus, the preparationcontained approximately 0.5 mg/ml of pure peptidoglutaminase II.

The peptidoglutaminase activity was determined by measuring the ammoniaformed during hydrolysis of γ-carboxyamide ofN-tert-Butoxycarbonyl-Gln-Pro (N-t-BOC-Gln-Pro; SIGMA No. B-4403) usingthe Boehringer-Mannheim kit for ammonia determination (Cat. No.1112732). In this kit, ammonia is measured by determination of theconsumption of NADH by glutamate dehydrogenase, and blanks without theaddition of N-t-BOC-Gln-Pro were also applied in order to subtract theeffect of other NADH consuming enzymes.

A total of 200 mg of wheat gluten protein was added to 9 ml of boilingwater and after cooling, the pH was adjusted to 7.0. Then 250 μl of thepeptidoglutaminase II preparation (PEP) described above was added. Theglutamate/aspartate specific protease (SP446) described in Example 16was added in an amount of 0.04 AU/g protein, and FLAVOURZYME™ describedin Example 16 was added in an amount of 20 LAPU/g protein.

Hydrolysis was allowed to proceed without pH adjustment for 18 hours at50° C. Controls without the addition of peptidoglutarninase were alsorun. The hydrolysates were centrifuged and glutamate was measured asdescribed in Example 16. The DH was determined as described in Example15.

The results as shown below in Table 5 demonstrated that hydrolysis withthe peptidoglutaminase preparation increased the DH as well as therelease of glutamate.

TABLE 5 DH Glutamate Hydrolysis % mg/l Minus PEP 40 131 Plus PEP 43 171

Deposit of Biological Materials

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli DH5α pMWR52 NRRLB-21682 Apr. 18, 1997

13 1 4280 DNA Aspergillus 1 aatttcctca ctcatccttc tatccaccgc caaaatgaaggccgctaccc tcctctctct 60 tctgagcgtt accggactcg tcgccgctgc tccagctggcaacggtacgt atcctgaacg 120 acaatgtaag acgcttgact gatgattagt aggcccagctggtggaatca tcgaccgcga 180 tcttcccgtc cctgtccctg gactccctac caagggtctccctattgttg acggattgac 240 tggcggcaat aagggtggcg agaagcctgg aagcaaggttactcctcgtg aagaccctac 300 cggcagcgcc cctgatggca agggcaatga tggccccgacggtgatctta ccggacgtcc 360 cggtcaaggg ggtcttgaca accctttcga tctccctactccagagcttc ctcccgtcaa 420 gcttcctggc ggacttgacg gtggcaaggg cggtctcggccttcgtcgtc gtggcagccc 480 agtagacggt ctccctgtcg ttgggcctgt tgttggtggtgttctaggtg gcggtggtgc 540 tggcagtggt gctggtgcca agggtggtgc tggtagtggtaccgttgggc gtcgtggcag 600 cccagtagac ggtctccctg ttgttgggcc tgttgttggtggtgtcctag gtggcggtgg 660 tgctggcagt ggtgctggtg ccaagggtgg tgctggtagtggtaccccta agcgccgtga 720 cggtccagtg gacggtgttc ctgtcgttgg agagcttgctgaaggtgcta ctggaggtct 780 tctaggtggt gatgctggtt ctgctgatgc tgctggtgctgatgctggtg ctgatgctgg 840 tgctggtgct ggtgggcaat agtctaacaa gggctttacggcatcaatgt gaggttatcc 900 aacatccatc cttggtggcc attcgtaaat agcaacaaagaggggtggta cttggtcgcg 960 atgtcattgc tcctgcgatt gaagctagcg attcctgtatgtacaataat tttaagcacg 1020 cttggttcca tactgtttct tcactggttt ttggatattttttcacttat tgaatcttgt 1080 agtagtccag cttctcatgg ttagacacgg gataaccccccaatagcatc atctgcaggt 1140 ttgatgttgc aatggtcaag ttttgtctta aattatgtacgagtcttggg ttacaccgct 1200 agaagctttg ccaccaatga agctgtagct tgtccaacggctatcagcgg ttttttttat 1260 gagaatcttg gcaggatagg aaaagttggt ggtggtgaaggagctaatgc aggaggtgga 1320 gtgactgata agacgcgatt tctgcgggga aaaagaaaaaggaccaattt atgggactat 1380 ttatttaaac gggaagtctt caattccgct tcgccagccatcccttgatt cgagctgaac 1440 tcggggtttt ttccaccatg aaggtacgtc aattccactgattaaacatt atttgttaca 1500 tacactccat cattgagtca attataatta acacctcataattcagtact ccaagcttct 1560 gctgctcctg gtcagtgtgg tccaggccct ggatgtgcctcggaaaccac acgcgcccac 1620 cggagaaggc agtaagcgtc tcaccttcaa tgagaccgtagtcaagcaag caattacgcc 1680 gacctctcgc tcggtgcaat ggctctcggg cgcagaggatggatcctacg tgtacgcggc 1740 ggaagacggc agtctcacca tcgagaacat cgtcaccaacgagtcacgca cgctcatccc 1800 tgcggacaag attccgacag ggaaggaagc gttcaattactggatccatc ccgacttgtc 1860 gtcggtgctg tgggcgtcca accacaccaa gcagtatcggcattcgttct ttgccgatta 1920 ttacgtccag gatgtggagt cactcaagtc cgtgcccctgatgcccgatc aggaaggtga 1980 tattcaatat gcccaatgga gccccgtggg caataccatcgcttttgttc gcgagaatga 2040 cctttatgtc tgggataatg gtaccgttac tcgcattactgatgatggtg gccccgacat 2100 gttccacggc gtgccggact ggatctatga agaggagatcctcggcgatc gctacgcgtt 2160 gtggttctcg ccagatggtg aatatctggc ttacttgagcttcaatgaga ctggggttcc 2220 gacctacacc gttcagtatt atatggataa ccaagagatcgctccggcgt atccatggga 2280 gctgaagata aggtatccca aggtgtcgca gacgaatccgaccgtgacgt tgagtctgct 2340 taacatcgct agcaaggagg tgaagcaggc gccgatcgacgcgttcgagt caactgactt 2400 gatcattggc gaggttgctt ggctcactga tactcacaccaccgttgctg ctaaggcgtt 2460 caaccgtgtc caggaccagc aaaaggtcgt cgcggtcgatactgcctcga acaaggctac 2520 tgtcatcagc gaccgagatg ggaccgatgg atggctcgataaccttcttt caatgaagta 2580 tattggccct atcaagccgt ccgacaagga tgcctactacatcgacatct ctgaccattc 2640 gggatgggcg catctgtatc tcttccccgt ttcgggcggcgaacctatcc cactaaccaa 2700 aggcgactgg gaggtcacgt ctattctgag tattgatcaggaacgccagt tggtgtacta 2760 cctgtcgact caacaccaca gcaccgagcg ccatctctactccgtctcct attccacgtt 2820 tgcggtcacc ccgctcgtcg acgacaccgt tgccgcgtactggtctgctt ccttctccgc 2880 gaactcgggc tactacatcc tcacatacgg aggcccagacgtaccctacc aggaactcta 2940 cacgaccaac agtaccaaac cactccgcac aatcaccgacaacgccaaag tactcgagca 3000 aatcaaggac tatgcattgc ccaacatcac ctacttcgagcttcccctcc cctccggaga 3060 aaccctcaat gtgatgcagc gcttaccccc cgggttctccccggataaga agtaccccat 3120 acttttcacc ccatacggcg gcccaggcgc ccaagaagtgaccaagagat ggcaagccct 3180 gaatttcaag gcctatgtcg cctccgacag cgaactcgagtacgtaacct ggactgtcga 3240 caaccgcggc acaggtttca aaggacgcaa gttccgctccgccgtcacgc gccaactcgg 3300 cctcctcgaa gcagaagacc agatctacgc cgcgcaacaggcggccaaca tcccctggat 3360 cgatgcagac cacatcggca tctggggctg gagtttcggaggctacttga ccagcaaggt 3420 cctggagaag gacagcggtg ctttcacatt aggagtcatcaccgcccctg tttctgactg 3480 gcgtttctac gactcaatgt acacggagcg ctacatgaagaccctctcga ccaatgagga 3540 gggctacgag accagcgccg tccgcaagac tgacgggttcaagaacgtcg agggcggatt 3600 cttgatccag cacggaacgg gcgacgataa cgtccatttccagaactcgg ctgcgctggt 3660 ggatctcctg atgggcgatg gcgtctctcc tgagaagctccattcgcaat ggttcacaga 3720 ctcagaccac ggaatcagct accatggtgg cggcgtgttcctgtacaagc aactggcccg 3780 gaagctctac caggagaaga accgacagac gcaggtgctgatgcaccagt ggactaagaa 3840 ggacttggag gagtagaagc ggcacatcat tcattcattttaaagcgact ggctacacat 3900 agcatacata gcaattgata cttcgtattt taccctccccacagccacga ccatcaccca 3960 ttggcgcaaa attctccccg caccataaac tagcgcgacgaggctgaaaa tctgccagaa 4020 atctacttaa agctcgtgtt ggcccagtcc ctcacaacccaaaccatccc aagtaaacaa 4080 aaccaaaaaa aaatcccata gaaaatggcc gacatccccactcaacagtc caaatcacaa 4140 ccctccccac caaatccgta acaatcaccc cgcaacgagcgaccatcgtt cgcgagatac 4200 acacctccat ccaggtatgc acataccacc tcacctgaccatccaaccct acttacagtc 4260 aacgtaaact aacaaaattc 4280 2 771 PRTAspergillus oryzae 2 Met Lys Tyr Ser Lys Leu Leu Leu Leu Leu Val Ser ValVal Gln Ala 1 5 10 15 Leu Asp Val Pro Arg Lys Pro His Ala Pro Thr GlyGlu Gly Ser Lys 20 25 30 Arg Leu Thr Phe Asn Glu Thr Val Val Lys Gln AlaIle Thr Pro Thr 35 40 45 Ser Arg Ser Val Gln Trp Leu Ser Gly Ala Glu AspGly Ser Tyr Val 50 55 60 Tyr Ala Ala Glu Asp Gly Ser Leu Thr Ile Glu AsnIle Val Thr Asn 65 70 75 80 Glu Ser Arg Thr Leu Ile Pro Ala Asp Lys IlePro Thr Gly Lys Glu 85 90 95 Ala Phe Asn Tyr Trp Ile His Pro Asp Leu SerSer Val Leu Trp Ala 100 105 110 Ser Asn His Thr Lys Gln Tyr Arg His SerPhe Phe Ala Asp Tyr Tyr 115 120 125 Val Gln Asp Val Glu Ser Leu Lys SerVal Pro Leu Met Pro Asp Gln 130 135 140 Glu Gly Asp Ile Gln Tyr Ala GlnTrp Ser Pro Val Gly Asn Thr Ile 145 150 155 160 Ala Phe Val Arg Glu AsnAsp Leu Tyr Val Trp Asp Asn Gly Thr Val 165 170 175 Thr Arg Ile Thr AspAsp Gly Gly Pro Asp Met Phe His Gly Val Pro 180 185 190 Asp Trp Ile TyrGlu Glu Glu Ile Leu Gly Asp Arg Tyr Ala Leu Trp 195 200 205 Phe Ser ProAsp Gly Glu Tyr Leu Ala Tyr Leu Ser Phe Asn Glu Thr 210 215 220 Gly ValPro Thr Tyr Thr Val Gln Tyr Tyr Met Asp Asn Gln Glu Ile 225 230 235 240Ala Pro Ala Tyr Pro Trp Glu Leu Lys Ile Arg Tyr Pro Lys Val Ser 245 250255 Gln Thr Asn Pro Thr Val Thr Leu Ser Leu Leu Asn Ile Ala Ser Lys 260265 270 Glu Val Lys Gln Ala Pro Ile Asp Ala Phe Glu Ser Thr Asp Leu Ile275 280 285 Ile Gly Glu Val Ala Trp Leu Thr Asp Thr His Thr Thr Val AlaAla 290 295 300 Lys Ala Phe Asn Arg Val Gln Asp Gln Gln Lys Val Val AlaVal Asp 305 310 315 320 Thr Ala Ser Asn Lys Ala Thr Val Ile Ser Asp ArgAsp Gly Thr Asp 325 330 335 Gly Trp Leu Asp Asn Leu Leu Ser Met Lys TyrIle Gly Pro Ile Lys 340 345 350 Pro Ser Asp Lys Asp Ala Tyr Tyr Ile AspIle Ser Asp His Ser Gly 355 360 365 Trp Ala His Leu Tyr Leu Phe Pro ValSer Gly Gly Glu Pro Ile Pro 370 375 380 Leu Thr Lys Gly Asp Trp Glu ValThr Ser Ile Leu Ser Ile Asp Gln 385 390 395 400 Glu Arg Gln Leu Val TyrTyr Leu Ser Thr Gln His His Ser Thr Glu 405 410 415 Arg His Leu Tyr SerVal Ser Tyr Ser Thr Phe Ala Val Thr Pro Leu 420 425 430 Val Asp Asp ThrVal Ala Ala Tyr Trp Ser Ala Ser Phe Ser Ala Asn 435 440 445 Ser Gly TyrTyr Ile Leu Thr Tyr Gly Gly Pro Asp Val Pro Tyr Gln 450 455 460 Glu LeuTyr Thr Thr Asn Ser Thr Lys Pro Leu Arg Thr Ile Thr Asp 465 470 475 480Asn Ala Lys Val Leu Glu Gln Ile Lys Asp Tyr Ala Leu Pro Asn Ile 485 490495 Thr Tyr Phe Glu Leu Pro Leu Pro Ser Gly Glu Thr Leu Asn Val Met 500505 510 Gln Arg Leu Pro Pro Gly Phe Ser Pro Asp Lys Lys Tyr Pro Ile Leu515 520 525 Phe Thr Pro Tyr Gly Gly Pro Gly Ala Gln Glu Val Thr Lys ArgTrp 530 535 540 Gln Ala Leu Asn Phe Lys Ala Tyr Val Ala Ser Asp Ser GluLeu Glu 545 550 555 560 Tyr Val Thr Trp Thr Val Asp Asn Arg Gly Thr GlyPhe Lys Gly Arg 565 570 575 Lys Phe Arg Ser Ala Val Thr Arg Gln Leu GlyLeu Leu Glu Ala Glu 580 585 590 Asp Gln Ile Tyr Ala Ala Gln Gln Ala AlaAsn Ile Pro Trp Ile Asp 595 600 605 Ala Asp His Ile Gly Ile Trp Gly TrpSer Phe Gly Gly Tyr Leu Thr 610 615 620 Ser Lys Val Leu Glu Lys Asp SerGly Ala Phe Thr Leu Gly Val Ile 625 630 635 640 Thr Ala Pro Val Ser AspTrp Arg Phe Tyr Asp Ser Met Tyr Thr Glu 645 650 655 Arg Tyr Met Lys ThrLeu Ser Thr Asn Glu Glu Gly Tyr Glu Thr Ser 660 665 670 Ala Val Arg LysThr Asp Gly Phe Lys Asn Val Glu Gly Gly Phe Leu 675 680 685 Ile Gln HisGly Thr Gly Asp Asp Asn Val His Phe Gln Asn Ser Ala 690 695 700 Ala LeuVal Asp Leu Leu Met Gly Asp Gly Val Ser Pro Glu Lys Leu 705 710 715 720His Ser Gln Trp Phe Thr Asp Ser Asp His Gly Ile Ser Tyr His Gly 725 730735 Gly Gly Val Phe Leu Tyr Lys Gln Leu Ala Arg Lys Leu Tyr Gln Glu 740745 750 Lys Asn Arg Gln Thr Gln Val Leu Met His Gln Trp Thr Lys Lys Asp755 760 765 Leu Glu Glu 770 3 20 PRT Aspergillus oryzae Xaa = Anyaminoacid 3 Xaa Glu Gly Ser Lys Arg Leu Thr Phe Xaa Glu Thr Val Val LysGln 1 5 10 15 Ala Ile Thr Pro 20 4 22 PRT Aspergillus oryzae 4 Gln ArgLeu Pro Pro Gly Phe Ser Pro Asp Lys Lys Tyr Pro Ile Leu 1 5 10 15 PheThr Pro Tyr Gly Gly 20 5 7 PRT Aspergillus oryzae 5 Lys Tyr Ile Gly ProIle Lys 1 5 6 7 PRT Aspergillus oryzae 6 Gly Glu Gly Ser Lys Arg Leu 1 57 8 PRT Aspergillus oryzae Xaa = Any aminoacid 7 Xaa Pro Ile Leu Phe ThrPro Tyr 1 5 8 16 PRT Aspergillus oryzae Xaa = Any aminoacid 8 Xaa ValPro Leu Met Pro Asp Gln Gln Gly Asp Ile Gln Tyr Ala Gln 1 5 10 15 9 21PRT Aspergillus oryzae 9 Gly Ala Tyr Thr Gly Gly Ile Thr Ile Thr Ala TyrGly Ala Arg Gly 1 5 10 15 Ala Arg Gly Ala Arg 20 10 32 PRT Aspergillusoryzae 10 Gly Gly Arg Thr Ala Tyr Thr Thr Tyr Thr Thr Arg Thr Cys IleGly 1 5 10 15 Gly Ile Ser Trp Arg Ala Ala Ile Cys Cys Ile Gly Gly IleGly Gly 20 25 30 11 931 PRT Saccharomyces cerevisiae 11 Met Ser Ala SerThr His Ser His Lys Arg Lys Asn Ser His Leu Phe 1 5 10 15 Pro Gln ArgLys Ser Ser Asn Ser Ser Met Asp Lys Pro Phe Phe Pro 20 25 30 Asn Asn AspSer Val Ala Asn Thr Asp Pro Gln Ser Asn Glu Asn Gly 35 40 45 His Thr IleAsn Glu Ile Arg Pro Thr Glu Ala Thr Ile Asp Val Thr 50 55 60 Asp Val ProGln Thr Pro Phe Leu Gln Glu Gln Tyr Ser Met Arg Pro 65 70 75 80 Arg ArgGlu Ser Phe Gln Phe Asn Asp Ile Glu Asn Gln His His Thr 85 90 95 His SerPhe Phe Ser Val Asn Lys Phe Asn Arg Arg Trp Gly Glu Trp 100 105 110 SerLeu Pro Glu Lys Arg Ser Tyr Val Leu Val Phe Thr Leu Ile Ala 115 120 125Leu Ser Val Leu Val Leu Leu Val Ile Leu Ile Pro Ser Lys Leu Leu 130 135140 Pro Thr Lys Ile Thr Arg Pro Lys Thr Ser Ala Gly Asp Ser Ser Leu 145150 155 160 Gly Lys Arg Ser Phe Ser Ile Glu Asn Val Leu Asn Gly Asp PheAla 165 170 175 Ile Pro Glu Asp Thr Phe His Phe Ile Asp Pro Pro Gln ArgLeu Leu 180 185 190 Gly Gln Asp Ser Asp Pro Gly Leu Tyr Phe Thr Thr LysGlu Ile Asp 195 200 205 Gly His Thr Asn Phe Ile Ala Lys Gln Leu Phe AspGlu Thr Phe Glu 210 215 220 Val Asn Leu Gly Gly Asn Arg Phe Leu Tyr GluGly Val Glu Phe Thr 225 230 235 240 Val Ser Thr Val Gln Ile Asn Tyr LysLeu Asp Lys Leu Ile Phe Gly 245 250 255 Thr Asn Leu Glu Ser Glu Phe ArgHis Ser Ser Lys Gly Phe Tyr Trp 260 265 270 Ile Lys Asp Leu Asn Thr GlyAsn Ile Glu Pro Ile Leu Pro Pro Glu 275 280 285 Lys Ser Asp Asp Asn TyrGlu Leu Gly Leu Ser Lys Leu Ser Tyr Ala 290 295 300 His Phe Ser Pro AlaTyr Asn Tyr Ile Tyr Phe Val Tyr Glu Asn Asn 305 310 315 320 Leu Phe LeuGln Gln Val Asn Ser Gly Val Ala Lys Lys Val Thr Glu 325 330 335 Asp GlySer Lys Asp Ile Phe Asn Ala Lys Pro Asp Trp Ile Tyr Glu 340 345 350 GluGlu Val Leu Ala Ser Asp Gln Ala Ile Trp Trp Ala Pro Asp Asp 355 360 365Ser Lys Ala Val Phe Ala Arg Phe Asn Asp Thr Ser Val Asp Asp Ile 370 375380 Arg Leu Asn Arg Tyr Thr Asn Met Asn Glu Ala Tyr Leu Ser Asp Thr 385390 395 400 Lys Ile Lys Tyr Pro Lys Pro Gly Phe Gln Asn Pro Gln Phe AspLeu 405 410 415 Phe Leu Val Asn Leu Gln Asn Gly Ile Ile Tyr Ser Ile AsnThr Gly 420 425 430 Gly Gln Lys Asp Ser Ile Leu Tyr Asn Gly Lys Trp IleSer Pro Asp 435 440 445 Thr Phe Arg Phe Glu Ile Thr Asp Arg Asn Ser LysIle Leu Asp Val 450 455 460 Lys Val Tyr Asp Ile Pro Ser Ser Gln Met LeuThr Val Arg Asn Thr 465 470 475 480 Asn Ser Asn Leu Phe Asn Gly Trp IleGlu Lys Thr Lys Asp Ile Leu 485 490 495 Ser Ile Pro Pro Lys Pro Glu LeuLys Arg Met Asp Tyr Gly Tyr Ile 500 505 510 Asp Ile His Ala Asp Ser ArgGly Phe Ser His Leu Phe Tyr Tyr Pro 515 520 525 Thr Val Phe Ala Lys GluPro Ile Gln Leu Thr Lys Gly Asn Trp Glu 530 535 540 Val Thr Gly Asn GlyIle Val Gly Tyr Glu Tyr Glu Thr Asp Thr Ile 545 550 555 560 Phe Phe ThrAla Asn Glu Ile Gly Val Met Ser Gln His Leu Tyr Ser 565 570 575 Ile SerLeu Thr Asp Ser Thr Thr Gln Asn Thr Phe Gln Ser Leu Gln 580 585 590 AsnPro Ser Asp Lys Tyr Asp Phe Tyr Asp Phe Glu Leu Ser Ser Ser 595 600 605Ala Arg Tyr Ala Ile Ser Lys Lys Leu Gly Pro Asp Thr Pro Ile Lys 610 615620 Val Ala Gly Pro Leu Thr Arg Val Leu Asn Val Ala Glu Ile His Asp 625630 635 640 Asp Ser Ile Leu Gln Leu Thr Lys Asp Glu Lys Phe Lys Glu LysIle 645 650 655 Lys Asn Tyr Asp Leu Pro Ile Thr Ser Tyr Lys Thr Met ValLeu Asp 660 665 670 Asp Gly Val Glu Ile Asn Tyr Ile Glu Ile Lys Pro AlaAsn Leu Asn 675 680 685 Pro Lys Lys Lys Tyr Pro Ile Leu Val Asn Ile TyrGly Gly Pro Gly 690 695 700 Ser Gln Thr Phe Thr Thr Lys Ser Ser Leu AlaPhe Glu Gln Ala Val 705 710 715 720 Val Ser Gly Leu Asp Val Ile Val LeuGln Ile Glu Pro Arg Gly Thr 725 730 735 Gly Gly Lys Gly Trp Ser Phe ArgSer Trp Ala Arg Glu Lys Leu Gly 740 745 750 Tyr Trp Glu Pro Arg Asp IleThr Glu Val Thr Lys Lys Phe Ile Gln 755 760 765 Arg Asn Ser Gln His IleAsp Glu Ser Lys Ile Ala Ile Trp Gly Trp 770 775 780 Ser Tyr Gly Gly PheThr Ser Leu Lys Thr Val Glu Leu Asp Asn Gly 785 790 795 800 Asp Thr PheLys Tyr Ala Met Ala Val Ala Pro Val Thr Asn Trp Thr 805 810 815 Leu TyrAsp Ser Val Tyr Thr Glu Arg Tyr Met Asn Gln Pro Ser Glu 820 825 830 AsnHis Glu Gly Tyr Phe Glu Val Ser Thr Ile Gln Asn Phe Lys Ser 835 840 845Phe Glu Ser Leu Lys Arg Leu Phe Ile Val His Gly Thr Phe Asp Asp 850 855860 Asn Val His Ile Gln Asn Thr Phe Arg Leu Val Asp Gln Leu Asn Leu 865870 875 880 Leu Gly Leu Thr Asn Tyr Asp Met His Ile Phe Pro Asp Ser AspHis 885 890 895 Ser Ile Arg Tyr His Asn Ala Gln Arg Ile Val Phe Gln LysLeu Tyr 900 905 910 Tyr Trp Leu Arg Asp Ala Phe Ala Glu Arg Phe Asp AsnThr Glu Val 915 920 925 Leu His Leu 930 12 36 DNA Aspergillus oryzae 12gatttaaatc accatgaagg tacgtcaatt ccactg 36 13 36 DNA Aspergillus oryzae13 gttaattaat ctactcctcc aagtccttct tagtcc 36

What is claimed is:
 1. An isolated polypeptide having dipeptidylaminopeptidase activity, selected from the group consisting of: (a) apolypeptide having an amino acid sequence which has at least 95%identity with amino acids 17 to 771 of SEQ ID NO:2; (b) a polypeptidewhich is encoded by a nucleic acid sequence which hybridizes under highstringency conditions with (i) nucleotides 49 to 2396 of SEQ ID NO. 1,or (ii) the cDNA sequence corresponding to nucleotides 49 to 2396 of SEQID NO. 1, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing with 2×SSC, 0.2% SDS at 65° C.; and (c) a fragment of (a) or (b)wherein the fragment has dipeptidyl aminopeptidase activity.
 2. Thepolypeptie of claim 1 comprising an amino acid sequence which has atleast 95% identity with amino acias 17 to 771 of SEQ ID NO:2.
 3. Thepolypeptide of claim 2, comprsing an amino acid sequence which has atleast 97% identity with amino acids 17 to 771 of SEQ ID NO:2.
 4. Thepolypeptide of cliam 1, comprising tne amino acid sequence of SEQ IDNO:2.
 5. The potypeptide of claim 1, consisting of tne amino acidsequence of SEQ ID NO:2 or a fragment thereof having dipeptidylaminopeptidase activity.
 6. The polypeptide of claim 5, consisting ofthe amino acid sequence of SEQ ID NO:2.
 7. The polypeptide of claim 6,consisting of amino acids 17 to 771 of SEQ ID NO:2.
 8. The polypeptideof claim 2, which is obtained from an Aspergillus strain.
 9. Thepolypeptide of claim 8, which is obtained from an Aspergillus oryzaestrain.
 10. The polypeptide of claim 1, which is encoded by a nucleicacid sequence which hybridizes under high stringency conditions with (i)nucleotides 49 to 2396 of SEQ ID NO. 1, or (ii) the cDNA sequencecorresponding to nuclotides 49 to 2396 of SEQ ID NO. 1, wherein highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing with 2×SSC, 0.2% SDS at 65° C.11. The polypeptide of claim 10, which is obtained from an Aspergillusstrain.
 12. The polypeptide of claim 12, which is obtained from anAspergillus oryzae strain.
 13. The polypeptide of claim 1, which isencoded by the nucleic acid sequence contained in plasmia pMWR52 whichis contained in E. coli NRRL B-21682.
 14. The polypeptide of claim 1,which acts synergistically with an aminopeptidase to hydrolyze apolypeptide.
 15. An isolated polypeptide having dipeptidylaminopeptidase activity from an Aspergillus strain, having the followingphysicochemical properties: (a) a pH optimum at about pH 8.7 determinedafter incubation for 5 minutes at ambient temperature in the presence of2.9 mM Ala-Pro-para-nitroanilide; (b) a temperature stability of 90% ormore, relative to initial activity, after incubation for 20 minutes at65° C. pH 7.5 in the absence of substrate, wherein remaining activitywas determined with 2.9 mM Ala-Pro-para-nitroanilide in 50 mM sodiumphosphate pH 7.5; (c) activity towards Xaa-Pro-para-nitroanilide orXaa-Ala-para-nitroanilide at ambient temperature in 50 mM sodiumphosphate pH 7.5, wherein Xaa is selected from the group consisting ofAla, Arg, Asp, Gly, and Val; and (d) a molecular weight of about 93-96kDa by SDS-PAGE.
 16. The polypeptide of claim 15, which is obtained froman Aspergillus oryzae strain.
 17. A flavor-improving compositioncomprising a polypeptide of claim 1 and a suitable carrier.
 18. Apre-mix for a dough comprising a polypeptide of claim 1 and a bakingingredient.
 19. An isolated nucleic acid sequence encoding a polypeptidehaving dipeptidyl aminopeptidase activity, selected from the groupconsisting of: (a) a nucleic acid sequence encoding a polypeptide havingan amino acid sequence which has at least 95% identity with amino acids17 to 771 of SEQ ID NO:2; (b) a nucleic acid sequence which hybridizesunder high stringency conditions with (i) nucleotides 49 to 2396 of SEQID NO. 1, or (ii) the cDNA sequence corresponding to nucleotides 49 to2396 of SEQ ID NO. 1, wherein high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatures salmon sperm DNA, and 50% formamide, andwashing with 2×SSC, 0.2% SDS at 65° C.; and (c) a nucleic acid fragmentof (a) or (b), which encodes a polypeptide having dipeptidylaminopeptidase activity.
 20. The nucleic acid sequenc of claim 19, whichis obtained from an Aspergillus strain.
 21. The nucleic acid sequence ofclaim 20, which is obtained from an Aspergillus oryzae strain.
 22. Thenucleic acid sequence of claim 19, which is encoded by the nucleic acidsequence contained in plasmid pMWR52 which is contained in E. coli NRRLB-21682.
 23. A nucleic acid construct comprising the nucleic acidsequence of claim 19 operably linked to one or more control sequenceswnich direct tne production of the poypeptide in a suitable expressionhost.
 24. A recombinant expression vector comprising the nucleic acidconstruct of claim 23, a promoter, and transcriptional and translationalstop signals.
 25. A recombinant host cell comprising the nucleic acidconstruct of claim
 23. 26. A method for producing a polypeptie havingdipeptidyl aminopeptidase activity comprising (a) cultivating the hostcell of claim 25 under conditions suitable for production of thepolypeptide; and (b) recovering the polypeptide.